Effects of cococonut water and simvastatin in the treatment of sepsis and hemorrhagic shock in rats1

PURPOSE: To evaluate the effects of modified coconut water as fluid of resuscitation combined with simvastatin in hemorrhagic shock and sepsis model in rats. METHODS: Four groups of Wistar rats with hemorrhagic shock and abdominal sepsis were studied (n=8/group). Rats were bled and maintained at a mean blood pressure 35mmHg for 60min. They were then resuscitated with: 1) saline 0.9%; 2) coconut water+3% NaCl; 3) coconut water+NaCl 3%+simvastatin microemulsion (10 mg/kg i.v.; 4) normal coconut water. At 8h post-resuscitation, blood and lungs were collected for exams. RESULTS: Clinical scores, TNF-α, IL-1β, liver/kidney proof levels, and lung injury were significantly reduced in coconut water+NaCl 3%+simvastatin group treated rats, comparing with the other resuscitation treatments. CONCLUSIONS: Resuscitation with coconut water with Nacl 3%+simvastatin had a significant beneficial effect on downregulating cytokines and decreasing lung injury in a rat model of abdominal sepsis and hemorrhagic shock. We also demonstrated that coconut water with Nacl 3%+simvastatin administration clearly made liver and kidney function better and improved clinical score.


Introduction
Pathophysiology of sepsis is related to an imbalance between anti-inflammatory and pro-inflammatory substances that will mediate a response to damage body tissues 1 . Despite intensive treatment with aggressive resuscitation, blood pressure control and adequate supply of oxygen, patients with sepsis often persist showing signs of tissue hypoperfusion, which can lead to acidosis and eventually multiple organ failure [2][3][4] . Researchers have shown that sepsis is characterized by decreased velocity of microcirculatory flow, increased flow heterogeneity, increasing vascular stasis and decreasing perfused capillaries 5,6 . Failure in the microcirculation flow results in shunting of blood from the tissues, leading to deficit in oxygen required for normal cell metabolism 7,8 .
Systemic inflammatory mediators and endotoxins disrupt intracellular connections and the signals used by endothelial cells, acting as a unified system. This disruption can result in altered distribution of blood flow in tissues 9 .
The hemorrhagic shock and consequent tissue hypoperfusion lead to a reduction of cell oxygenation, metabolic acidosis and hypothermia 10 . The reduction of O2 supply leads to increased anaerobic metabolism and reduction of ATP levels and intracellular calcium, which promote significant changes in cellular function by stimulating the release of pro-inflammatory cytokines. These, in turn, may alter the immune function of macrophages and lymphocytes, causing immunosuppression and increased risk of infection 11 .
Some works have studied the composition of coconut water (CW). Santoso et al. 12 described the presence of vitamins, sugars, organic acids, fatty acids, amino acids, minerals and electrolytes in the coconut (Cocus nucifera L.) water. Aleixo et al 13 determined their selenium content using atomic absorption spectrometry. Another important feature of CW is its antioxidant ability [14][15][16] . Trace elements and metals in small dosages were determined, leading to believe that CW has potential for use in intravenous hydration or parenteral nutrition solutions supplement 17 . Campbell-Falck et al. 18 reported a successful case of intravenous hydration in the Salomon Islands and described its use for this purpose during the II World War.
The electrolyte composition of the 6 months CW resembles more the intracellular fluid then the extracellular, and consists mainly of potassium, calcium, magnesium and chlorine.
Sodium is found in much lower concentration than in human plasma 19 . Although CW has already been described as a viable solution for resuscitation, reports of its i.v. use are scarce 20,21 , with the information that it does not interfere in hemostasis mechanisms 22 .
Statins inhibit HMG-CoA reductase and have been extensively studied for the prevention of atherosclerosis, inflammatory disease of the vascular endothelium, whose pathogenesis has similarities with the pathogenesis of sepse 23 .
Although the effects of statins were initially assigned only to treat hyperlipid levels, many other benefits came to be considered and studied. Statins exert multiple effects on various cells for a number of mechanisms. These pleiotropic effects have been described as antiinflammatory, modifying the interactions between the endothelium and leukocytes 24,25 . Additionally, statins modulate the signaling of inflammatory cells, which in turn reduces the release of cytokines and proteins of acute phase of sepsis 26 , and other important antioxidant effects 27 .
The ideal solution for volume replacement in situations of sepsis associated with hemorrhagic shock is not yet well established. In this protocol we studied the effects of modified CW solution, combined with intravenous simvastatin for the volume replacement in rats submitted to a double challenge: sepsis and hemorrhagic shock. These situations are common and deleterious in surgical practice, with high morbidity and mortality.
This study aimed to examine the effects of the modified CW combined with simvastatin as fluid resuscitation in hemorrhagic shock and abdominal sepsis model in rats.

Methods
The institutional Ethics Committee on Animal Use approved this protocol, which was performed at the Nucleus of  We recorded the volume of collected blood to calculate the fluid volume to be reinfused. MAP was monitored through an Invasive Blood Pressure Monitor. Hemorrhagic shock was maintained for 60 minutes.

Abdominal sepsis and study groups design
After 60 min of shock, for the induction of the sepsis rats

Fluid resuscitation
Coconut water (CW) was obtained from 6 months coconut (Cocus nucifera L.) specimens (time from the inflorescence to harvesting the fruits), using sterile technique. CW was harvested immediately before administration to animals and modified appropriately to achieve the 3% NaCl level. We measured the CW pH with pHmeter (Micronal, São Paulo, Brazil), (mean pH 5.8). After that, pH was adjusted to the value 7.4 by using sodium bicarbonate 10%. Sodium content of CW was adjusted to 3% immediately prior to intravenous infusion, by adding precalculated volume of NaCl 10%. Saline solution 0.9% was from B. Braun, Rio de Janeiro, Brazil. We used infusion pump (B.

Laboratory parameters determination
The surviving animals after 18 hours of observation were again anesthetized and blood was harvested for dosages. Serum was separated by centrifugation at 3000 rpm and stored at -40°C for subsequent dosing. Plasma levels of TNF-α and IL-1β were determined using quantitative enzyme linked immunosorbent assay (ELISA) kits, according to manufacturer recomendations (PeproTech, USA). We determined aspartate aminotransferase

Histological evaluation of lung injury
The lungs were removed and immersed in 10% buffered formalin for 48 h and then embedded in paraffin. 4μm sections were stained with hematoxylin and eosin. Pulmonary architecture was evaluated by optical microscopy with BX50 microscope equipped with digital camera DS30 (Olympus, Japan). Two random tissue sections of four different lungs from each group were examined.
Lung injury scores were quantified by an investigator blinded to the treatment groups using previous published criteria (Table 1), which gives an overall score between 0 and one 29 .   Serum levels of inflammatory cytokines TNF-α and IL-1β were measured in all groups, and were found to be significantly lower in the coconut water +simvastatin-treated group. Although all groups showed a significant difference (p<0.05) compared to the saline treated group, the best results were found in the coconut water +simvastatin-treated group rats (Table 2). More studies in this area are needed to decipher the mechanism of this particular issue. As for biochemical dosages, the rats treated with CW + Na3% and simvastatin had lower levels of C-reactive protein, AST, ALT, urea and creatinine then in the other groups, with statistically significant differences ( Table 3). The same happened with the white blood cell count, where the total leukocytes and neutrophils values of the animals treated with CW + Na3% and simvastatin, were significantly lower than in the other groups. However, as shown in Table 4, the percentage of eosinophils was significantly higher in rats treated with CW + Na3% and simvastatin (p<0.05).  The fluorescence signal intensity (SI) of the lungs was measures in Saline and CW Na3%+ simvastatin groups.
Fluorescent images (Figure 2) showed signal intensity markedly higher in the saline group rats compared to animals in the CW Na3%+ simvastatin group. In Table 5 is shown that corresponding quantitative SI of lungs fluorescence in saline group animals were significantly higher compared to the CW Na3%+ simvastatin group after intravenous injection of 10 mg/kg ICG.

Histopathology
As shown in Figure 3, histopathological changes and lung injury were evident in lung tissues from rats with CLP-induced sepsis and shock treated with saline, compared to rats treated with coconut water. Light microscopic analysis of lung tissue from animals with sepsis and shock, treated with saline 0.9% showed thickened alveolar septum with increased cellularity. These animals developed increased neutrophils and a prominent increase in mononuclear and interstitial cells in alveolar septum. Alveolar edema was also present in this group. It is clear that histological damage and leukocytes infiltration were ameliorated after coconut water + Na3% + simvastatin treatment and was accompanied with a declined histology scores. There was no increase in neutrophils in alveolar septum, and minor cells or edema in the alveolar spaces ( Figure 3). So, lung biopsy, done in animals at 18 h after treatment with coconut water 3% (Na) + simvastatin, showed minor changes in lung architecture by light microscopy. Histopathologic scores are summarized in Table 6.

Discussion
The hemorrhagic shock causes tissue damage and increases hypoxia and proinflammatory activity. A rapid resuscitation with adequate fluids is essential to minimize damage and improve tissue perfusion. The present study examined an alternative to reduce the damage caused by hypovolemia using normal and modified coconut water, which showed positive results compared to resuscitation with 0.9% saline. In the present study, we compared the effects 0.9% saline, modified coconut water (Na 3%) and water coconut 3% + Na + simvastatin, and normal coconut water on serum cytokines, biochemical and white blood cell count in rodent model with hemorrhagic shock and abdominal sepsis. Experimental models of sepsis and hemorrhagic shock has often been employed in other estudies [30][31][32] . In the present study, we found that treatment of rats with sepsis and shock with coconut water modified with sodium 3% + simvastatin reduced serum cytokines, biochemical parameters and peripheral leukocytes. This reduction was statistically higher than the other fluids replacement treatments.
Simvastatin has shown immunomodulatory effects, independent of significant reduction in hyperlipidemia 35 .
These pleiotropic effects include anti-inflammatory action 36  In addition, CW has significant amount of antioxidants that protect the body against oxidative stress caused by hypoxia inherent to hemorrhagic shock 16 .
The indocyanine green (ICG) has been extensively used for decades in the fields of ophthalmology for retinal imaging and cardiology to study cardiac output. ICG has a well documented safety profile 40,41 . We used a higher dose for rodents as described for patients in order to compensate the short blood half life of ICG in rodents (1.5 -2.3 min) as opposed to patients (3 -4 min) 42,43 .
The relatively high ICG dose provided a prolonged contrast enhancement and allowed sequential imaging of lungs without additional contrast injection.
Our results showed that inflamed and normal lung tissues differed significantly in normalized fluorescence. On the basis of the different temporal behavior of fluorescence intensity in septic rats, it is more likely that vasodilation in the inflamed lungs was present and new microvessels were formed, leading to a faster inflow of ICG contrast agent. Fluorescence imaging is limited to superficial organs and tissues, because the strong scattering and absorption of light in biologic tissue restricts the penetration depth of photons to a few millimeters. As fluorescence in vivo was not detectable with our device in lungs of rats in vivo, we decided to perform ex vivo imaging, and representative images were recorded in inflamed lungs. A way to further improve the specificity of this method is to use disease-specific contrast agents in combination of a marker for neovascularization.

Conclusions
The volume replacement with coconut water modified with Na 3%, combined with simvastatin had a positive influence in the treatment of rats with sepsis + hemorrhagic shock model.
The findings may have significant therapeutically implications in the clinical setting.