Heart and systemic effects of statin pretreatment in a rat model of abdominal sepsis

PURPOSE: To evaluate the heart and the Tc-99m-sestamibi biodistribution after statin pretreatment in a rat model of abdominal sepsis. METHODS: Twenty-four Wistar rats were randomly distributed into four groups (n=6 per group): 1) sepsis with simvastatin treatment, 2) sepsis with vehicle, 3) sham control with simvastatin and 4) sham control with vehicle. 24 hours after cecal ligation and puncture rats received 1.0MBq of Tc-99m–sestamibi i.v. 30min after, animals were euthanized for ex-vivo tissue counting and myocardium histological analysis. RESULTS: Myocardial histologic alterations were not detected 24 hours post-sepsis. There was significantly increased cardiac Tc-99msestamibi activity in the sepsis group with simvastatin treatment (1.9±0.3%ID/g, p<0.001) in comparison to the sepsis group+vehicle (1.0±0.2%ID/g), control sham group+ simvastatin (1.2±0.3%ID/g) and control sham group (1.3±0.2%ID/g). Significant Tc-99msestamibi activity in liver, kidney and lungs was also detected in the sepsis group treated with simvastatinin comparison to the other groups. CONCLUSIONS: Statin treatment altered the biodistribution of Tc-99m-sestamibi with increased cardiac and solid organ activity in rats with abdominal sepsis, while no impact on controls. Increased myocardial tracer activity may be a result of a possible protection effect due to increased tissue perfusion mediated by statins.


Introduction
Sepsis is the leading cause of death in critically ill patients 1 , mainly as a result of multiple organ failure. Cardiac dysfunction is one of the complications of sepsis, being capable of increasing the mortality by 70% 2 .
The mechanisms of sepsis-induced cardiac dysfunction have been studied extensively 3 . In recent years, several drugs have been tested for prevention and treatment of sepsis, with discouraging results. Some studies, however, are showing benefits with HMG-CoA reductase inhibitor (enzyme responsible for the biosynthesis of cholesterol). Currently it is consolidated that statins (HMG-CoA reductase inhibitors), reduce mortality in patients with atherosclerosis 4 , reduce the volume of atherosclerotic plaque inflammation and control mechanisms associated with the atheroma genesis 5 . Other effects of statins that are increasingly being reported are the imunomodulation effects 6 . It has been also been established that statins can increase the expression of nitric oxide (NO) 7 .
In sepsis induced cardiac dysfunction, a protective role for statins has been suggested. Using a circulatory shock model induced by lipopolysaccharide (LPS) in guinea pigs, Giuoti-Paiva et al. 8 evaluated the production of NO and the cardiovascular response to the infusion of phenylephrine in simvastatin treated or non-treated groups. NO levels increased significantly two hours after the injection of LPS compared to the control group. In the group pre-treated with simvastatin the NO levels were significantly reduced. LPS injection produced prolonged hypotension in the experimental group; pretreatment with simvastatin did not prevent this hypotensive effect, but the response to phenylephrine was restored in the statin treated group.
Another factor responsible for septic cardiac dysfunction is the poor distribution of regional blood flow induced by sepsis.
Poor blood flow distribution can contribute to myocardial dysfunction, generating areas of ischemia. It is assumed that statins also have vasoprotective effects. In a study by Liuba et al. 9   Tc-sestamibi is a radiopharmaceutical that is widely used for myocardial perfusion imaging. The kinetics of this radiopharmaceutical in the myocardium and its biodistribution has been reported in several experimental models 10,11 . In the presence of irreversible myocardial injury, mitochondrial membranes are depolarized by changing the uptake of 99m Tc-sestamibi 12 . The uptake of 99m Tc-sestamibi is dependent on the myocardial tissue viability and regional blood flow 13 .
Assuming that cardiac dysfunction can be caused or aggravated by sepsis, and that the inhibition of inflammation is one of many pleiotropic effects of statins, we tested the following hypothesis: pretreatment with simvastatin have a protection effect on the heart, and can possibly have an impact on the cardiac uptake and biodistribution of 99m Tc-sestamibi using an experimental model of abdominal sepsis in rats.

Sepsis induction
Animals were fasted 12h before the experiment and anesthetized with intramuscular injection of 0.1 mL/100g weight, of a solution prepared with 1.0 mL of ketamine (50mg/mL) and 1.0 mL of xilazine (20mg/mL). They breathed spontaneously throughout the procedures. After shaving, the abdominal skin was disinfected with 70% alcohol. All procedures were performed under sterile conditions. A 3 cm midline laparotomy was performed and cecal ligation and puncture (CLP) was performed. The cecum was exposed, ligated with cotton 3-0, one cm distally to the ileocecal valve to avoid intestinal obstruction. Four punctures were performed with a 22-gauge needle, squeezed gently to force out a small amount of feces, and then it was returned to the abdominal cavity. The abdominal incision was closed with 4-0 nylon sutures. Midline laparotomy (3 cm) and gentle manipulation of cecum was performed in the sham rats. Pain medication (meperidine 10 mg/body weight) and volume support (NaCl 0.9%, 0.05 mL/g body weight) were applied subcutaneously immediately after the induction of sepsis and every 12 hours thereafter.
Twelve animals were treated orally with simvastatin and twelve with 0.9% saline. Six animals with sepsis and six sham were injected daily with oral suspension of simvastatin 10 mg/kg/ day, (gavage) for three days prior to induction of peritonitis and 2h before the CLP. The other rats received oral 1 ml of 0.9% saline.
After 24 hours postoperative observation the animals were anesthetized, and a dose of 1.0 MBq of 99m Tc-sestamibi was injected intravenously. The injected dose (ID) was calculated as the difference between the measured radioactivity in the seringe before and after injection, using a curiemeter (Capintec CRC-25R). Thirty minutes after injection, animals were euthanized, and the heart, lung, kidney and liver were resected. The samples were quickly washed in saline, After obtaining the tissue biodistribution measurements, the fresh hearts were cut and washed in running water to enable rapid and uniform action of the fixative solution. Then the samples were fixed in 10% buffered formalin for 48 hours and processed for 18 hours in an automatic tissue processor, using Leica equipment TP 1020, German.
Prior to embedding in paraffin, the left ventricles of fixed hearts were cut with punch (6 mm diameter), for standardization of samples.
Histological sections were obtained with microtome Leica RM 2125 RTS, 03 microns thick. The fixed specimens were stained with hematoxylin/eosin for morphological analysis by optical microscopy, using the CX41 microscope (Olympus, Tokyo, Japan). Sections were examined in high magnification power fields (x400) to determine the presence of adherent and infiltrating neutrophils, eosinophils, basophils, monocytes, lymphocytes and platelets. The total number of cells was analyzed in six fields for each heart expressed in cells per square millimeter. The quantitative analysis was performed using a video-assisted software (Image ProPlus 6.0, Media Cyber).

Data analysis
All data were presented as mean±standard deviation and compared by ANOVA and Tukey test. The difference between the means was considered statistically significant when p <0.05.

Results
All animals survived the experiments. Contraction necrosis or interstitial fibrosis was not seen in any of the evaluated hearts. Interstitial edema, mononuclear infiltrate, myocytolysis and tissue hemorrhage were found, but no differences were detected when comparing sepsis and sham groups, with and without treatment with simvastatin.
The average time for resection of organs for weighing on a precision scale and measurements of the 99m Tc-sestamibi activities was 10 minutes per animal. Table 1 show the percentage of the injected activity per gram of tissue (% ID/g) detected in the heart, lung, kidney and liver, in each group.
The highest %ID of 99m Tc-sestamibi per gram of tissue was detected in the heart and kidney both in sham and in the sepsis groups ( Table 1). The lowest %ID of 99m Tc-sestamibi per gram of tissue was detected in the lungs in all groups (Table 1).
There were no significant differences in the %ID/g 99m Tcsestamibi of per gram of tissue in the heart, liver, kidney and lung among sham groups treated with simvastatin and those treated with saline ( Table 1). The %ID/g of the liver and the kidney was significantly higher in the sepsis groups, when compared with the sham groups ( Table 1). The %ID/g in the heart and lung was lower in the sepsis group treated with saline when compared to sham, however with no significant difference ( Table 1). The %ID/g of the heart was significantly higher in the sepsis group pretreated with simvastatin than in the sepsis group treated with saline. The myocardium activity was also significantly higher than in sham groups treated with simvastatin and with saline. There was also a significant %ID/g of tissue in the lung, kidney and liver in sepsis groups treated with simvastatin, when compared with the control groups, and when compared to the sepsis group treated with saline (Table 1).

Organs
%ID/g per group p-value (

Discussion
Recently, our group showed that simvastatin had significant anti-inflammatory effect in rats with abdominal sepsis, using the CLP model. The results showed that TNF-α, IL-1β and IL-6 values in septic group previously treated with simvastatin were significantly lower than in the untreated sepsis group. The same occurred in total leukocytes and neutrophils 14 . In another study, our group showed that simvastatin also had important anti-inflammatory action in the abdominal sepsis in diabetic rats.
Simvastatin reduced mortality in diabetic rats. Serum levels of TNF-α, IL-1β, IL-6, C-reactive protein, procalcitonin, leukocytes, and neutrophils were significantly lower in diabetic and nondiabetic rats with sepsis treated with simvastatin, than in the group treated with saline 15 . In this study, cardiac and systemic effects of simvastatin pretreatment were analyzed in septic rats, using 99m Tcsestamibi as a specific substrate to assess biodistribution in the heart, liver, kidney and lung. The exact mechanism of cellular uptake of 99m Tc-sestamibi is still unclear. Due to the lipophilic nature of the 99m Tc-sestamibi cation, it is apparently distributed across biological membranes in response to transmembrane potential 12  Overall, the results of our study showed that CLPinduced abdominal sepsis was associated with increased retention of 99m Tc-sestamibi in the heart and in the liver, kidney and lung samples, especially in sepsis group pretreated with simvastatin.
Wang et al. 19 showed similar results evaluating the activity of P-glycoprotein through the biodistribution of 99m Tc-sestamibi in endotoxemic rats. These findings could possibly reflect low excretion and distribution of 99m Tc-sestamibi, secondary the lower activity of P-glycoprotein during sepsis. The 99m Tc-sestamibi is eliminated from the body primarily through active secretion mediated by the activity of P-glycoprotein. As such, high levels of also showed that changes in blood concentration could not fully explain changes in accumulation in vital organs.
Importantly, no significant correlation between serum levels of 99m Tc-sestamibi and accumulation were found in organs both in the group treated with LPS and control groups. It is therefore less likely that there is statin-induced liver and kidney dysfunction leading to increased 99mTc-sestamibi cardiac retention in the sepsis group with simvastatin. Also, different from Wang et al. 19 , our results showed that the whole heart distribution of 99mTcsestamibi in sepsis pretreated with simvastatin was significantly higher than in the saline treated and sham groups. The mechanism for the increased cardiac distribution of 99mTc-sestamibi is unclear. One possible hypothesis is that simvastain could possibly be enhancing the intracellular accumulation of 99mTc-sestamibi due to presumable amplification of P-glycoprotein function inhibition. Mendes et al. 21 19 detected mdr1a mRNA in heart, but at lower levels than those in the liver and kidney.
However, mdr1a levels were significantly depressed in the heart of LPS-treated mice, but this decrease caused only slight changes in the cardiac biodistribution of 99m Tc-sestamibi. High affinity of heart tissue by 99m Tc-sestamibi and the relative low activity of P-glycoprotein may have contributed to these findings. Therefore, changes in P-glycoprotein activity induced by inflammation are unlikely to cause significant impact of cardiac uptake of 99m Tcsestamibi. This finding is consistent with the findings of our study.
Our results showed no significant changes in the levels of 99m Tcsestamibi in sepsis group treated with saline compared to the sham group. However, the whole heart distribution of 99m Tc-sestamibi in sepsis rats pretreated with simvastatin was significantly higher than in the saline treated and sham groups.
Another hypothesis to explain the increased cardiac biodistribution of 99m Tc-sestamibi in the sepsis group previously treated with simvastatin is possibly due to increased tissue perfusion secondary to coronary vasodilation induced by increasing concentration of nitric oxide under the action of statins 7 . Merx et al. 23 showed an improved survival of rats with sepsis, justifying that this was due to cardiac and hemodynamic stability after treatment with statins. Their study also showed that the cardiac and hemodynamic stability was associated not only to sinvastatin, but with other statins as weel. The mechanisms were related to the better susceptibility to stimulation of nitric oxide synthase and reduction of leukocyte endothelial adhesion in animals treated with statins. To better assess this hypothesis experiments with microspheres labeled with raioactive isotopes or quantitative myocardium perfusion PET kinetic studies using ammonia N-13 or labeled water O-15 would be necessary. Therefore, a proven statin protection effect could have a major impact in sepsis induced cardiac dysfunction treatment.

Conclusions
Statin treatment altered the biodistribution of Tc-99msestamibi with increased cardiac and solid organ activity in rats with abdominal sepsis, while no impact on controls. Increased myocardial tracer activity may be a result of a possible protection effect due to increased tissue perfusion mediated by statins.