We showed that the inclusion of variables easily measurables 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 with the established MSS, the above-mentioned variables might be considered as a new score for the evaluation of sepsis in experimental models (Table 1).
Table 1
Adapted Murine Sepsis Score (A-MSS)
Score | 0 | 1 | 2 | 3 | 4 |
Appearance | Coat is smooth | Patches of hair piloerected | Majority of back is piloerected | Piloerection may or may not be present, mouse appears “puffy” | Piloerection may or may not be present, mouse appears emaciated |
Level of consciousness | Mouse is active | Mouse is active but avoids standing upright | Mouse activity is noticeably slowed. The mouse is still ambulant | Activity is impaired. Mouse only moves when provoked, movements have a tremor | Activity severely impaired.Remains stationary when provoked, with possible tremor |
Activity | Normal amount of activity. Mouse is any of: eating, drinking, climbing, running, fighting | Slightly suppressed activity. Mouse is moving around bottom of cage | Suppressed activity. Mouse is stationary with occasional investigative movements | No activity | No activity. Mouse experiencing tremors, particularly in the hind legs |
Response to stimulus | Mouse responds immediately to auditory stimulus or touch | Slow or no response to auditory stimulus; strong response to touch (moves to escape) | No response to auditory stimulus; moderate response to touch (moves a few steps) | No response to auditory stimulus; mild response to touch (no locomotion) | No response to auditory stimulus. Little or no response to touch. Cannot right itself if pushed over |
Eyes | Open | Eyes not fully open, possibly with secretions | Eyes at least half closed, possibly with secretions | Eyes half closed or more, possibly with secretions | Eyes closed or milky |
Respiration rate | Normal, rapid mouse respiration | Slightly decreased respiration (rate not quantifiable by eye) | Moderately reduced respiration (rate at the upper range of quantifying by eye) | Severely reduced respiration (rate easily countable by eye, 0.5 s between breaths) | Extremely reduced respiration (> 1 s between breaths) |
Respiration quality | Normal | Brief periods of laboured breathing | Laboured, no gasping | Laboured with intermittent gasps | Gasping |
Rectal temperature (ºC) | 36–38 | > 38 | < 36 - ≥ 35 | < 35 - ≥ 34 | < 34 |
Casual blood glucose (mg/dL) | ≥148 | ≤ 148 - ≥122 | < 122 - ≥58 | <58 - >40 | ≤ 40 |
Relative body weight loss (%) | 0–5 | 5–10 | 10–15 | 15–20 | > 20 |
Sepsis is a complex disease that requires a complex form to diagnose it successfully. [25]. 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. [26]. 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 [27] [27]. These dysfunctions cause tissue damage and impair brain function [28]. 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 [11], but using the A-MSS allows the assessment of autonomic insufficiency evaluating the temperature (Fig. 1c).
Neurohypophysial dysfunction promotes decreased hepatic gluconeogenesis-promoting adrenocortical hormones and liver and muscle glycogenolysis, causing a decrease in glycemia [29]. Furthermore, LPS is able to decrease the activity of hepatic and renal enzyme phosphoenolpyruvate carboxykinase (PEPCK) promoting gluconeogenesis, and hypoglycemia [30], as observed in Fig. 1b.
Sepsis impairs neuroendocrine regulation by neuronal dysfunction impairing the secretion of vasopressor hormones [31], accompanied by the desensitization of receptors for vasoconstriction [32]. Also, LPS and cytokines induce the nitric oxide production by macrophages, neutrophils and monocytes [32]. Decreased cardiac output and blood pressure may be associated with hypothermia (Fig. 1c) [11, 37], reinforcing the need to measure body temperature in sepsis mice models.
A catabolic condition leads to a reduction in muscle mass [38] is related to increased hospitalization period and mortality [14]. This metabolic effect is also verified in an experimental sepsis model [9, 10, 39], and the results found in our study (Fig. 1d) may be associated with muscle atrophy and lipid catabolism [40]. Also, leukocytes release interleukin-1 beta, which has a direct effect on appetite inhibition and food intake, reflecting in body weight loss [41].
The platelet function disorders play a crucial role in the pathophysiology of sepsis, with prognostic accuracy [42]. LPS and cytokine endotoxemia (TNF-α, tumor necrosis factor-alpha, IL-8, IL-15) stimulate endothelium, monocytes, neutrophils and basophils to secrete platelet-activating factor (PAF) [42]. PAF triggers platelet aggregation with the formation of microthrombi, which, together with leukocyte-induced hemophagocytosis [43], may result in decreased total platelet count (thrombocytopenia) and its relative rate [44]. 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 [45], as observed in our study (Figure S2c). It has been proposed that MPV indicates severity in just after 72 hours [46] and that the evaluation of MPV is insufficient to predict the worsening of sepsis due to peritonitis with gram-negative bacteria [15]. The dysfunction in the platelet count can also be observed by analyzing the ratios between the MPV and platelet count (MPV/CP), representing the risk of microthrombi formation [47]. The platelet is a predictor of worsening of sepsis, specifically when it accuses the systemic infection with gram-negative bacteria [15], as we observed in our study (Figure S2d).
Immunosuppression observed by decreasing lymphocyte and monocytes leads to a decrease in total leukocytes (Figure S3b,c,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 [48]. Neutrophils are immune cells of the first line of defense against bacterial infection and may suffer exacerbated apoptosis in severe sepsis [15].
Studies suggest the application of NLR and PLR as inflammatory biomarkers [15]. PLR and NLR elevation is a prognostic marker of lethality in patients with peritonitis [15], as well as in an animal models [49], similarly to Figure S3e,f. Also, platelet and immune biomarkers may indicate etiological agents [15]. 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 (Figure S3a-f).