Adapted Murine Sepsis Score: Improving the Research in Experimental Sepsis Mouse Model

The Murine Sepsis Score (MSS) is used to assess the severity of sepsis in rats and mice based on observational characteristics. The quantitative variables of glycemia, body weight, and temperature are predictors of severity in experimental models of sepsis. Therefore, our study sought to adapt the MSS with the same variables to indicate earlier the severity of the disease in murine models of the disease. Sepsis mice presented hypoglycemia, weight loss, and hypothermia. Therefore, these variables were included in the Adapted Murine Sepsis Score (A-MSS). The A-MASS presented 100% specificity and 87.5% sensibility been able to differentiate the early sepsis symptoms and its severity. The A-MSS allows an early and more complete diagnosis of sepsis in mice and might be considered as a procedure to improve the analysis of systemic sepsis dysfunction in murine experimental models.


Introduction
Sepsis is a systemic infection characterized by multiple organ dysfunction and high mortality rates. Therefore, studies investigating the pathophysiology in sepsis are required [1,2], and the use of experimental mouse models has been considered a fundamental approach [3], due to the physiological similarities between human and mice, including a great genotypic homology in terms of hormonal, cell signaling, receptors, immune system, and oxidative and cell stress responses [4]. The bacteria-induced sepsis in animal models has similar characteristics found in septic patients [5,6] due to the gram-negative bacteria membrane endotoxins like lipopolysaccharide (LPS) and lipoteichoic acid (LTA) from gram-positive bacteria [7].
The pathogen-associated molecular patterns (PAMPS) are recognized by cell surface receptors, for example, Tolllike receptors, expressed by the human and animal leukocytes. In this way, LPS is recognized by the host cell defense by TRL4, whereas TLR2 receptors recognize LTA. Both molecular interactions induce the activation of the nuclear kappa B transcription factor (NF-κB), which initiates the synthesis of several proinflammatory cytokines. Also, these PAMPS may interact with platelets and endothelial tissue, adding microvascular complications that have an essential role in the inflammation and systemic dysfunction in sepsis [8].
One of the leading infectious focus in sepsis is abdominal origin. Staphylococcus aureus and Escherichia coli bacteria are commensals of the mouse gastrointestinal tract and can be found in the feces of these animals [5,9]. One of the leading infectious focus in sepsis is abdominal origin. Staphylococcus aureus and Escherichia coli bacteria are commensals of the mouse gastrointestinal tract and can be found in the feces of these animals [5,9]. Therefore, an experimental model of sepsis is peritonitis induction by the intraperitoneal administration of the autogenous fecal solution. From this procedure, sepsis is recognized by an application of Murine Sepsis Score (MSS) that consists of observing and categorizing the level of consciousness, activity, behavior, response to stimuli, respiratory rate, and quality of breathing movements [10]. However, it is possible to observe a decreased glycemic levels, core temperature, and body weight loss if we follow up animals for 24 hours [11,12], but these variables have not been included in the MSS protocol [10].
Patients and animals with sepsis may present hypothermia [13,14], hypoglycemia [11,15], body weight loss [16], and hematological alterations [17]. Also, early identification of sepsis is a well-known, relevant procedure to allow successful treatment of the disease [18]. Therefore, herein, we proposed an adaptation in the MSS (A-MSS) to provide a score that identifies sepsis in an experimental model in the initial stages.

Experimental
Design. The animals were divided into two experimental groups: control (n = 7) and sepsis (n = 8). A 20% fecal solution (200 mg/mL) was prepared with fresh stool in 0.9% NaCl, and it was administered at a dose of 5 μL/g (1 mg/g, i.p.) in the sepsis group, while control animals received 0.9% NaCl [12]. The animals were monitored for glycemia, blood count, rectal temperature, and MSS (Table S1), before (time zero) and at 4, 12, and 24 hours after sepsis induction.
2.4. Procedures Details. Glycemia was measured by distal tail puncture (~5 μL) using Optium Xceed® glucometer. Rectal temperature was measured with a digital thermometer, and body weight was measured using a semianalytical balance. For hematological analysis, blood was collected (0, 4, 12, and 24 h) by caudal puncture (10 μL). Samples were diluted  Hematological strains were performed on the slide for differential leukocyte count, stained with a panoptic type [20].

Statistical
Analysis. Data were analyzed using the Kolmogorov-Smirnov test. The results in which the evaluation was performed over time were verified by two-way ANOVA (time × treatment) followed by Bonferroni posttest. Spearman or Pearson correlation test was performed. The potential diagnosis of A-MSS within four hours after sepsis induction was tested by ROC curve. The cutoff point for A-MSS diagnosis was determined by observing the most appropriate balance between sensitivity and specificity in the ROC curve [21]. The significance level of 5% (P < 0:05) was considered.

Results
The animals submitted to sepsis presented increased MSS 12 h and 24 h after fecal solution administration (Figure 1(a)). Also, animals with sepsis showed a decrease in glycemia, rectal temperature, and body weight (Figures 1(b)-1(d)). It was possible to verify that sepsis induces alterations in appearance, level of consciousness, activity, stimulus-response, eye aspect, and respiratory rate and quality (Supplementary Figure S1). The neutrophil count increased in 12 h after sepsis induction, then returning to basal level in the next 12 hours (Figure 3(a)). At 24 h, we found a decrease in white blood cell count, including a decrease in lymphocyte and monocyte count (Figures 3(d), 3(b), and 3(c), respectively).
These alterations in immune cells, as well as observed in the platelets, impact in the neutrophil-lymphocyte ratio (NLR), which increased at 12 h together with the platelet to lymphocyte ratio (PLR) in 24 h (Figures 3(e) and 3(f), respectively).
A strong negative correlation (r = −0:79) was observed between MSS and glycemia 24 hours after sepsis induction. Also, a moderate negative correlation was observed between MSS and body temperature (r = −0:61) and weight loss (r = −0:52) in the same time-point (Figures 4(a)-4(c)). Therefore, we proposed the A-MSS which was able to report sepsis within four hours of the sepsis induction ( Figure 4(d)). When this score was evaluated from the ROC curve in this period, it was found that it can be used as a diagnostic standard (Figure 4(e)), from an A-MSS value of 3.5, obtaining a sensitivity of 87.5% and specificity of 100%.

BioMed Research International
The proposed A-MSS is detailed in Table 1 Figure S2D). There is no statistically significant correlation between A-MSS and platelet to lymphocyte ratio (P > 0:05) and also between A-MSS and MPV/PC ratio (P > 0:05).

Discussion
We showed that the inclusion of variables easily measurable as glycemia, temperature, and body weight in the MSS may improve the research in sepsis mice model. Our proposal of A-MSS represents a sum of observations, and together  (Table 1). Sepsis is a complex disease that requires a complex form to diagnose it successfully [22]. The A-MSS added parameters to the MSS, which allowed an indirect assessment of the cardiovascular (temperature) and metabolic systems (glycemia and body weight), which are essential for the prognosis in sepsis in animals and humans. In humans, the diagnosis is made by measures of neurological, cardiovascular, respiratory, renal, hepatic, and platelet dysfunction, by the reproducible Sequential Organ Failure Assessment (SOFA) score [23]. We believe that A-MSS approximates the clinical variables evaluated in SOFA for the early diagnosis of sepsis in animal models. The central nervous system dysfunction is mainly characterized by septic encephalopathy, followed by autonomic  failure [24]. These dysfunctions cause tissue damage and impair brain function [25]. In our study, these neurological effects were reflected in the impairment of the level of consciousness and activity in the sepsis group ( Figure S1A, C). The neurological symptoms can be identified in the first hours by MSS [13], but using the A-MSS allows the assessment of autonomic insufficiency evaluating the temperature (Figure 1(c)). Neurohypophysial dysfunction promotes decreased hepatic gluconeogenesis-promoting adrenocortical hormones and liver and muscle glycogenolysis, causing a decrease in glycemia [26]. Furthermore, LPS is able to decrease the activity of hepatic and renal enzyme phosphoenolpyruvate carboxykinase (PEPCK) promoting gluconeogenesis and hypoglycemia [27], as observed in Figure 1(b).
Sepsis impairs neuroendocrine regulation by neuronal dysfunction impairing the secretion of vasopressor hormones (NASCIMENTO et al., 2017), accompanied by the desensitization of receptors for vasoconstriction [28]. Also, LPS and cytokines induce the nitric oxide production by macrophages, neutrophils, and monocytes [28]. Decreased cardiac output and blood pressure may be associated with hypothermia (Figure 1(c)) [13,29], reinforcing the need to measure body temperature in sepsis mouse models.
A catabolic condition leads to a reduction in muscle mass [30] is related to increased hospitalization period and mortality [16]. This metabolic effect is also verified in an experimental sepsis model [11,12,31], and the results found in our study (Figure 1(d)) may be associated with muscle atrophy and lipid catabolism [32]. Also, leukocytes release interleukin-1 beta, which has a direct effect on appetite inhibition and food intake, reflecting in body weight loss [33].
The platelet function disorders play a crucial role in the pathophysiology of sepsis, with prognostic accuracy [34]. LPS and cytokine endotoxemia (tumor necrosis factoralpha (TNF-α), IL-8, IL-15) stimulate endothelium, BioMed Research International monocytes, neutrophils, and basophils to secrete plateletactivating factor (PAF) [34]. PAF triggers platelet aggregation with the formation of microthrombi, which, together with leukocyte-induced hemophagocytosis [35], may result in decreased total platelet count (thrombocytopenia) and its relative rate [36]. Regarding MPV, it is already known that the 24 h period is insufficient for the increase in MPV to indicate the severity in patients with sepsis [37], as observed in our study (Figure 2(c)). It has been proposed that MPV indicates severity in just after 72 hours [38] and that the evaluation of MPV is insufficient to predict the worsening of sepsis due to peritonitis with gram-negative bacteria [17]. The dysfunction in the platelet count can also be observed by analyzing the ratios between the MPV and platelet count (MPV/PC), representing the risk of microthrombi formation [39]. The MPV/PC is a predictor of worsening of sepsis, specifically when it accuses the systemic infection with gram-negative bacteria [17], as we observed in our study (Figure 2(d)). Immunosuppression observed by decreasing lymphocyte and monocytes leads to a decrease in total leukocytes (Figures 3(b)-3(d)). On the other hand, the bone marrow has a reservoir of neutrophils that are released into the circulation to combat infectious in the peritoneal cavity, as verified 12 h after sepsis induction [40]. Neutrophils are immune cells of the first line of defense against bacterial infection and may suffer exacerbated apoptosis in severe sepsis [17].
Studies suggest the application of NLR and PLR as inflammatory biomarkers [17]. PLR and NLR elevation is a prognostic marker of lethality in patients with peritonitis [17], as well as in an animal models [41], similarly to Figures 3(e) and 3(f). Also, platelet and immune biomarkers may indicate etiological agents [17]. Thus, the A-MSS proposed in our study was able to indicate sepsis in mice just after four hours, correlated with the 24-hour values of the biomarkers mentioned above: lymphocyte count, white blood cell count, platelet count, and NLR. These pieces of evidence allow proposing the A-MSS as a relevant tool for murine severe infectious diseases since it was able to predict the severity systemic dysfunction of the mouse sepsis model.

Conclusion
The A-MSS allows an early and more complete diagnosis of sepsis in mice and might be considered as a procedure to improve the analysis of systemic sepsis dysfunction in mice. The inclusion of new variables that can be directly measured represents the inclusion of objective criteria in a quite subjective exam to improve the accuracy of studies in mouse severe sepsis model.

Data Availability
All data will be available under request for authors.

Additional Points
Limitations. Our study did not use laboratory tests for sepsis evaluations such as bilirubin, creatinine, or lactate.

Consent
All authors approved the submitted and published versions.

Disclosure
A preprint version of this manuscript was also available in the Research Square online platform in the link https:// www.researchsquare.com/article/rs-64379/v1.

Conflicts of Interest
All authors declared that they have no conflict of interest.  Table S1: Murine Sepsis Score (MSS).

Authors' Contributions
Supplementary 2. Figure S1: individualized MSS components, appearance, level of consciousness, activity, response to stimulus, aspect of eyes, respiratory frequency, and respiratory quality (A-G) within 24 hours after induction of sepsis with 20% fecal solution (1 mg/g).
Supplementary 3. Figure S2: correlation between A-MSS four hours after induction and lymphocyte count, total leukocytes, platelets, and the ratios between neutrophils and lymphocytes (NLR), platelets and lymphocytes (PLR), and mean platelet volume and platelet count (MPV/PC) (A-F) within 24 hours after induction of sepsis with 20% fecal solution (1 mg/g).