Despite recent medical advances, the incidence of sepsis has increased, especially after data collection in regions with a lower Socio-demographic Index (SDI) . The lack of knowledge about the evolution of the disease and the difficulty in providing an early diagnosis contribute to increase this trend and the mortality of patients [28, 29]. Current therapeutic strategies for treating sepsis depend on antibiotics, fluid replacement, and symptomatic therapy . Although there is no specific treatment for sepsis, several experimental studies have proposed potential new drugs or adjuvant therapies. In the present report, we show that the use of simvastatin 4 days before and 10 days after CLP-surgery improved metabolic parameters and prevented CNS changes in sepsis survivor animals.
Here, we show that at 24 and 48 hours after septic induction there is a progressive increase in bacterial load in the spleen and mesenteric lymph node of septic animals, coinciding with the time points of high mortality. Furthermore, we observed that bacterial dissemination increases in parallel with the increase of pro-inflammatory cytokines in the plasma of these animals. Recent studies have shown similar results by correlating severity and mortality scores with bacterial dissemination and cytokine levels in different organs in a pneumonia-induced sepsis model . Although we did not adopt a score to assess sepsis severity, our animals showed typical signs, such as lethargy, piloerection, and tachypnea, which are parameters considered reliable to attest the severity of the disease [32, 33]. Forty-eight hours after sepsis induction, the animals had a higher bacterial load in organs studied, higher plasma levels of proinflammatory cytokines, and a higher mortality rate. Other studies using the CLP model showed similar results, demonstrating that this appears to be the critical time point of sepsis in Wistar rats [24, 34].
The severity of sepsis has been linked to plasma cytokine concentrations for a long time . Considering 24 and 48 hours as the period with the highest number of animal deaths, the action of simvastatin was analyzed by quantifying the plasma levels of cytokines. With the simvastatin administration, survival was 100% at 24 hours and 60% at 48 hours after sepsis induction, remaining unchanged up to 10 days. It is likely that this maintenance of survival rate is related to the considerable decrease in IL-1b and IL-6 levels observed at this time point in animals treated with simvastatin. One of the biggest challenges in controlling the progression of sepsis is to modulate the 'cytokine storm' in the early stages of the disease [36, 37]. Cytokines are often responsible for triggering the inflammatory cascade and production of mediators such as nitric oxide, chemokines, and free radicals, leading to organ dysfunction and death [38, 39]. Previous work has shown that this anti-inflammatory effect of simvastatin, by decreasing the overproduction of pro-inflammatory cytokines, was able to limit the release of other mediators, such as nitric oxide and reactive oxygen species, and to increase the survival of septic animals [18, 40]. This pleiotropic effect of statins is extremely relevant, considering that several clinical and preclinical studies have used plasma levels of cytokines as surrogates for diagnosis and prognosis of organ dysfunction and mortality [37, 41–44].
Metabolic responses to sepsis are often difficult to measure, as they depend on several factors, including the body nutritional status and hormonal aspects [45, 46]. Furthermore, the stage of disease during which the patient or animal is studied must be considered and may explain the variability of results reported in the literature [47, 48]. In this work, we used indirect calorimetry to assess the energy metabolism in sepsis survivor rats treated with simvastatin. Our septic animals evaluated 48 hours after CLP-surgery had considerably lower calorimetric parameters (RQ = 0.90) compared to treated animals (RQ = 1.02). This discrepancy in the RQ values indicates that the animals of this experimental groups consume different substrates as an energy source, since higher RQ values suggest a predominant use of carbohydrates as an energy substrate . Moreover, our results showed an increase in the measurement of EE and locomotor activity of septic animals treated with simvastatin compared to those not treated, suggesting a restoration of energy production. Taken together, these results lead us to infer that the simvastatin administration may have contributed to the production of a hypermetabolic state, necessary for the recovery of these animals. Several authors agree that the absence or premature terminus of a hypermetabolic state is associated with severe prognosis [48, 50, 51]. Higher mortality was observed in septic animals that remained in a hypometabolic state and were unable to transition from lipid utilization to carbohydrates . Similarly, preterminal patients have hypofunctional changes in metabolism, such as decreased oxygen consumption, triglyceride clearance, and hepatic mitochondrial activity [47, 52].
The inability to sustain a hypermetabolic state and the incapacity to transit to carbohydrate utilization is associated with a dysregulation of the energy metabolism caused by mitochondrial dysfunction . Thus, it is possible to infer that the improvement in metabolic parameters and the restoration of energy production is related to the antioxidant action of statins [54, 55]. Previous studies have shown that treatment with statins reduced glial activation and oxidative stress at 48 hours after sepsis induction [17, 18]. It is likely that this effect is related to the inhibition of the synthesis of isoprenoid compounds and prenylation of small G proteins. The occurrence of these events limits the production of inflammatory mediators and reactive oxygen species by negatively modulating the binding of these G proteins to the plasma membrane to trigger the cell signaling cascade [22, 56].
Here, we observed that sepsis survivor animals evaluated 10 days after CLP-surgery showed an increase in RQ and EE values, suggesting success in the transition from lipid substrate to carbohydrates and reaching the hypermetabolic state. However, these animals were unable to restore body weight over the 10 days of evaluation. In experimental sepsis, the reduction in food and water consumption, even temporarily for 2 – 3 days post-CLP, is sufficient to maintain the animals body weight below baseline values for 7 days . In our study, it is likely that the severity of sepsis has produced a decrease in the search for food with consequent fasting and triggering of the mechanisms that promote lipolysis [57, 58]. Although we have noticed an improvement in the horizontal movements of the untreated survivors, the number of vertical movements remained lower compared to the treated survivors. This can be explained by the attempt to minimize energy expenditure in face of foraging activities necessary for survival, inasmuch as vertical movements are costly [59, 60]. The restoration of energy production at early time points provided simvastatin-treated rats with greater locomotor activity at both time points evaluated, in particular, an increase in vertical movements, observed even after 10 days of septic induction.
Patients that develop severe sepsis and septic shock have organ dysfunction and high levels of plasma cytokines, which can lead to death. Even with early intervention, patients who survive this condition are likely to develop neurocognitive impairments . Cognitive deficits found in sepsis survivor animals are due to neurodegenerative processes associated with sustained neuroinflammation . In this study, we performed morphological assessments in the hippocampus and prefrontal cortex, which are brain regions responsible for memory and cognition. We observed that animals surviving sepsis showed morphological characteristics suggestive of astrocytic activation and apoptosis. In sepsis, alterations in blood-brain barrier (BBB) permeability allow peripheral inflammatory mediators to reach the CNS [61, 62]. These inflammatory mediators play a key role in sustained glial activation, which, in turn, contributes to the perpetuation of a neuroinflammatory environment with consequent cell death and cognitive impairment . Our sepsis survivor animals did not show alterations in the plasma levels of IL-1 and IL-6, demonstrating that, although peripheral inflammation has been resolved, glial activation persists up to 10 days after sepsis. Other authors have reported long-term brain dysfunction in surviving animals using the CLP model [15, 34, 63].
Modulating sustained glial activation appears to be critical for containing persistent neuroinflammation in sepsis survivors [64–67]. Here, animals treated with simvastatin showed less noticeable morphological alterations in both investigated brain regions. Previous studies from our laboratory observed a decrease in Iba-1 expression accompanied by a reduction in cytokines and cleaved caspase-3, and an increase in Bcl-2 in the brain of the simvastatin-treated animals. The likely mechanism proposed for this anti-inflammatory effect of simvastatin is its role in the NF-κB (nuclear factor-κappaB)/SIRT1 (silent information regulator 1) signaling pathway by inhibiting the M1 microglia phenotype [68–72]. Since this microglial activation induces the neurotoxic phenotype of astrocytes through the release of large amounts of cytokines, suppressing this activation may explain the decrease in astrogliosis observed in treated animals [73, 74].
In sepsis, although organ system dysfunction is more easily evaluated through routine laboratory tests, there are still no well-defined and practical biological markers in the CNS that can be targeted for therapeutic interventions. Therefore, studies that investigate the course of disease evolution and its relationship with the neuroinflammatory response are needed to clarify the brain dysfunction observed in sepsis survivors. Here we demonstrate that simvastatin administered 4 days before and 10 days after CLP-surgery improved metabolic parameters by sustaining a hypermetabolic state necessary for animal survival, reduced levels of pro-inflammatory cytokines, and prevented damage to areas responsible for memory and cognition of survivor rats. We believe that the pre-treatment was crucial, because in a pilot study we did not obtain satisfactory results when administering this drug only after septic induction. Other authors reported similar results using statins before and after sepsis . As statins are widely used by the world population, further studies are needed to consider not interrupting this drug during the treatment of sepsis and to assess its likely potential as adjuvant neuroprotective therapy.