Effect of Post-Treatment Fluvastatin for Hemorrhagic Shock in Rats

Background To investigate the biochemical, histologic, and immunologic effects of post-treatment use of uvastatin in cases of hemorrhagic shock in a rat model. Methods Experimental rats were randomly divided into four groups: (1) the control group received no drugs and did not undergo hemorrhagic shock (HS); (2) the control + statin group received uvastatin 1 mg/kg without HS; (3) the HS group received normal saline after HS; (4) the HS + statin group received uvastatin 1 mg/kg + normal saline after HS. HS was induced by femoral arterial catheter blood extraction of 30% of the total blood volume. Mean arterial pressure and heart rate were monitored for 2 h after starting blood withdrawal. Arterial blood gas, complete blood count, and serum cytokine levels were measured at baseline, 2 hrs after HS, and 48 hrs after resuscitation. Kidney, lung, and small intestines were removed for pathological examination 48 h after HS. properties, it can also exhibit pleiotropic effects such as endothelial function improvement, antioxidant properties, stabilization of atherosclerotic plaques, modulation of progenitor cells, suppression of inammatory responses, and enhancement of certain immunomodulatory cytokine actions.(21, 25, 26) A study by Lee, Lee (7) found that pretreatment with uvastatin resulted in a decrease in HS-induced serum TNF-α level and an increased IL-10 production. These results suggest that uvastatin reduced the production of proinammatory cytokines and increases the production of anti-inammatory cytokines, thereby preventing HS-associated multi-organ damage. Our study showed that serum TNF-α level tended to decrease in the HS + statin group compared to the HS group. However, pro-inammatory cytokines were not signicantly decreased in the HS + statin group. On the other hand, a study by Chen, Lee (5) revealed that uvastatin can increase plasma IL-10 production when given after the onset of sepsis in a rat model and may have therapeutic effects in treatment of LPS-induced shock. These results are consistent with our ndings. In this study, treatment with uvastatin after HS resulted in higher IL-10 production than in the HS-only group 2 hrs after HS. This result suggested that post-treatment with uvastatin elevates serum anti-inammatory cytokines. However, these immunomodulatory effects were somewhat weak compared to pre-treatment with uvastatin. In addition, cytokine prole differences had little effect on laboratory and other measured parameters in the HS + statin group. This may be associated with the therapeutic dosage or pharmacokinetics of uvastatin used in our study. A previous study on the effects of intravenous rosuvastatin for acute stroke showed that it provided protection at concentrations of 0.2 and 2.0 mg/kg, but not at 0.02 mg/kg,(27) which could have been a similar factor in our experiments. Moreover, the elimination half-life uvastatin, multiple


Abstract Background
To investigate the biochemical, histologic, and immunologic effects of post-treatment use of uvastatin in cases of hemorrhagic shock in a rat model.

Methods
Experimental rats were randomly divided into four groups: (1) the control group received no drugs and did not undergo hemorrhagic shock (HS); (2) the control + statin group received uvastatin 1 mg/kg without HS; (3) the HS group received normal saline after HS; (4) the HS + statin group received uvastatin 1 mg/kg + normal saline after HS. HS was induced by femoral arterial catheter blood extraction of 30% of the total blood volume. Mean arterial pressure and heart rate were monitored for 2 h after starting blood withdrawal. Arterial blood gas, complete blood count, and serum cytokine levels were measured at baseline, 2 hrs after HS, and 48 hrs after resuscitation. Kidney, lung, and small intestines were removed for pathological examination 48 h after HS.

Results
The HS and HS + statins groups showed reduced bicarbonate, base excess, and platelet counts, all of which differed signi cantly from values in the control and control + statin groups at the end of the resuscitation period.
The HS + statin group exhibited signi cantly elevated serum IL-10 2 hrs after resuscitation compared with the control group (P < 0.05). Except for IL-10, the group-time interaction was not signi cant for other cytokine pro les.

Conclusion
This study demonstrated that post-treatment with uvastatin increased anti-in ammatory cytokines IL-10 production and affected cytokine pro les in rats after HS.

Background
Despite advances in trauma care, the leading cause of trauma death is exsanguination resulting in hemorrhagic shock (HS), which is seen in up to 30% of cases.(1) HS may result in multiple organ dysfunction and eventual death due to reduced tissue perfusion and subsequent cellular hypoxia, which occurs through several in ammatory pathways including leukocyte activation and organ sequestration. The current view holds that a dysfunctional systemic in ammatory response syndrome is central to the pathogenesis of multiple organ failure (MOF).
Proin ammatory cytokine release after shock is central to the in ammatory response and has been described in animal models of hemorrhagic and traumatic shock. Overwhelming production of pro-in ammatory cytokines such as tumor necrosis factor-α (TNF-α) can lead to hemodynamic instability. If this process is not controlled by antiin ammatory cytokines such as interleukin-10 (IL-10), it can lead to multiple organ dysfunction and death after HS.
(2) HMG-CoA reductase inhibitors (statins) exhibit important immunomodulatory effects independent of any lipidlowering effect that they may elicit.(3) A novel lipid-independent mechanism of action for statins that is unrelated to their inhibitory potential on HMG-CoA reductase has recently been de ned. Some previous studies showed that statins may exert an anti-in ammatory effect by binding to a speci c site of lymphocyte function associated antigen-1 (LFA-1) on leukocytes and found that pre-treatment with uvastatin suppressed the release of tumor necrosis factor-α (TNF-α), increased interleukin-10 (IL-10) production, and decreased levels of organ injury markers in endotoxic shock in rats. (4,5) Furthermore, a post-treatment test giving uvastatin to mice appeared to prolong survival following cecal ligation and puncture (CLP)-induced sepsis. (6) Since HS can produce the same in ammatory effects as endotoxic shock after LPS, it is possible that uvastatin may also play a protective role in HS. Recent experimental studies showed that pre-treatment with uvastatin suppressed the release of TNF-α, increased IL-10 production, and decreased markers of organ injury after induction of HS in rats.
To the best of our knowledge, no previous studies have focused on the effects of post-treatment with uvastatin upon HS and the expression levels of multiple cytokines including IL-6, IL-10, IL-1β, IL-12p70, TNF-α, and IFN-γ. The aim of our study was to investigate the biochemical, histologic, and immunologic effects of post-treatment use of uvastatin in cases of HS in a rat model.

Subjects
Forty male Sprague-Dawley rats (weight, 295.7 ± 22.3; age, 8-9 weeks old; Charles-River, Montreal, Quebec, Canada) were used and housed at the university animal center in a controlled environment at 22 ± 2 °C with a 12hour light/dark cycle. The rats had unlimited access to food and water before the experiment.

Animal preparation
Animals were anesthetized with an intramuscular injection of 1 cc/kg tiletamine/zolazepam (Zoletil®, Virbac, France). After sterile left-groin dissection, a 24-gauge catheter was cannulated into the femoral artery to monitor blood pressure, sample blood, and withdraw blood. Another 24-gauge catheter was inserted into the femoral vein for uid infusion and intravenous drug administration. The surgical procedure was completed in 15 minutes, and the incision was less than 0.5 cm 2 in length, as small as possible. Heparin (80 IU/kg) was administered to prevent clotting in catheters. The femoral artery catheter was connected to a blood pressure transducer (FE221, AD Instruments, NSW, Australia), and systolic and diastolic blood pressure, mean arterial pressure (MAP), and heart rate were continuously recorded using PowerLab® (AD Instruments, NSW, Australia).

Experimental design
Animals were randomly divided into four groups. Group 1 was the control group, did not receive any drugs, and was not subjected to HS. Group 2 was the control + statin group, and these animals received 1 mg/kg uvastatin (Novartis Pharmaceuticals, Cambridge, MA, USA) without HS induction. Group 3 was subjected to HS and received normal saline after HS. Group 4 was the HS + statin group, and these subjects received 1 mg/kg uvastatin + normal saline after HS induction. Fluvastatins were given immediately after the rst blood sampling in group 2 or after 30 min of bleeding in group 4. The doses of drug given to rats were the same as a previous study.(7) Animals were randomized into equal-sized groups using a free online calculator offering random sample allocation. The mean weights of groups were: control, 293.7 ± 21.79 g; control + statin, 298.4 ± 14.97 g; HS, 298.8 ± 25.97 g; HS + statin, 291.0 ± 26.66 g. There were no statistically signi cant differences in body weight between the 4 groups. Blood was withdrawn through the femoral artery until MAP decreased to 60 mmHg. Rats were infused with normal saline to 3 times the volume of blood loss for uid resuscitation. The animals were moved to a cage after resuscitation. Rats were allowed free access to food and water after experiment completion. Acetaminophen (Children's Tylenol suspension1, Janssen, Seoul, Republic of Korea) was mixed with water to control pain in rats.
The total duration of this study was 48 hours and observation of rat health and behavior was performed every 12 h for 48 h to assess survival. Rats that survived to 48 h were euthanized by decapitation under terminal anesthesia.
The endpoint of our study included the survival of the rats until 48 hrs, so euthanasia was performed on live rats after that. Gross necropsy was performed on all rats, and all experimental processes are summarized in Fig. 1.

Blood sampling and measurements
Arterial blood gas such as pH, PO2, PCO2, HCO3, and excess base were measured at baseline, 2 hrs after HS, and 48 hrs after resuscitation. Complete blood count (CBC) including leukocyte count, hemoglobin, hematocrit, and platelet count were also measured at baseline, 2 hrs after administration of shock, and 48 hrs after resuscitation.

Histological examination
Lung, duodenum, and kidney specimens were removed immediately after sacri ce, xed overnight in 4% buffered formaldehyde, processed by standard methods, and stained with hematoxylin and eosin. A pathologist blinded to group enrollment subjected the lungs, duodenums, and kidneys to histopathological examination. All evaluations were made on ve elds per section and ve sections of lung, duodenum, and kidney.

Statistical analysis
Data are presented as mean ± standard error of the means (S.E.M.), mean ± standard deviation (S.D.) or median ± range, unless otherwise stated. Using repeated-measures ANOVA and Fisher's protected t-test, the signi cance of differences in measured values among groups was analyzed. SPSS 20.0 (SPSS, Inc., Chicago, IL, USA) was used to analyze the data. P-values less than 0.05 were considered statistically signi cant.

Arterial blood gas and complete blood count
No signi cant differences in laboratory values were found at baseline among the four groups (Tables 1). The HS group and HS + statin group had low bicarbonate, base excess, and platelet count, all of which differed signi cantly from values in the control and control + statin groups at the end of the resuscitation period. Baseline hemoglobin and hematocrit did not vary among the four groups, but they decreased signi cantly after blood loss at 2 hrs in the HS and HS + statin groups. Base excess and bicarbonate had recovered by 48 hrs after resuscitation in all groups, but the hemoglobin and hematocrit of the HS group and HS + statin group remained lower than those of the control group and control + statin group.
Plasma interleukin (IL)-1β, IL-6, IL-10, IL-12p70, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α Serial measurements of plasma interleukin (IL)-1β, IL-6, IL-10, IL-12p70, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α levels versus time plots are shown in Fig. 2. On repeated-measures ANOVA, the HS + statin group showed signi cantly elevated levels of serum IL-10 2 hrs after resuscitation compared with the other groups (*P < 0.05;). The HS group showed signi cantly elevated levels of serum TNF-α 2 hrs after resuscitation compared with the control group and control + statin group (*P < 0.05). The HS group and HS + statin group showed signi cantly elevated levels of serum TNF-α 24 hrs after resuscitation compared with the control group (*P < 0.05). Regarding other cytokine pro les, the group-time interaction was not signi cant among the four groups during the observation period.
Mean arterial pressure (MAP) and heart rate (HR) Initial MAP was higher than 140 mmHg in all groups. MAP decreased rapidly after controlled bleeding in the HS group and HS + statin group (P < 0.05; Fig. 3). After 2 hrs, the MAP of the HS group and HS + statin group stayed relatively was not different compared to the control group and control + statin group. HR signi cantly decreased after bleeding in the HS group and HS + statin group (P < 0.05; Fig. 3).

Total amount of uid resuscitation
Normal saline was infused during the resuscitation period in all HS groups. Three blood samplings were conducted for each animal, and 1 cc of blood was withdrawn for each laboratory test. And then, the same volume of normal saline was infused for animals in all groups. The mean amount of total infused normal saline was 11.8 ± 3.3 mL in the control group, 13.1 ± 1.7 mL in the control + statin group, 53.2 ± 6.1 mL in the HS group, and 50.9 ± 5.2 ml in the HS + statin group. There were no differences in the volume of normal saline between the HS group and HS + statin group.

Survival rate
All rats in the control group, control + statin group, and HS + statin group survived for 48 hrs, whereas one of 10 rats in the HS group did not recover from HS during the observation period and died. There was no difference in mortality between all group. The other rats were all euthanized 48 hours after study initiation.
Histopathology of kidney, lung, and small intestine There were no obvious histopathologic changes related to shock injury in the kidney, lung, and small intestine except for 1 case in the HS group. The most prominent histopathologic changes were observed in the lung tissue, including pulmonary hemorrhage and congestion. Neutrophils were also observed in lung tissue from the HS group (data not shown).

Discussion
This is the rst study to investigate the effects of uvastatin on the serum cytokine pro le of rats when administered immediately after HS. Our study found that the serum cytokines of the HS group and HS + statin group exhibited different pro les of cytokine activation compared with those of the control and control + statin groups. Especially, the HS + statin group exhibited an increased serum IL-10 level 2 hrs after resuscitation compared with the control group, while the HS group did not.
Cytokines have a variety of functions. Pro-in ammatory cytokine activation after shock is very important to the in ammatory response and has been well described in animal experiments of hemorrhagic shock. (8)(9)(10)(11) The proin ammatory role of TNF-α in the MOF condition has been established. (12,13) A major function of TNF-α at the cellular level is to up-regulate production of IL-1β by macrophages. TNF-α stimulates endothelial proliferation, neutrophil adhesion, free radical formation, and IL-6 production. The role of IL-6 in the in ammatory reaction remains unclear, though previous studies have supported both pro-in ammatory and anti-in ammatory roles for this cytokine. IL-10, which has the ability to suppress cytokines and inhibit effector functions of immune cells, has been suggested to play an important role in counter regulation of the early in ammatory response. (14,15) The proportion of IL-6 to IL-10 correlated well with the severity of injury in trauma patients in one clinical study. (16) During hemorrhage, in ammatory cytokines such as TNF-α and IL-10 play important and early roles in HS-induced organ damage. (17,18) Accordingly, there are several studies that attempt to suppress TNF-α, which is an important component of in ammation. Abraham, Jesmok (19) and Bahrami,Yao (20) found that hemorrhage and resuscitation-induced acute lung injury can be reduced through TNF-α monoclonal antibody therapy. In another study, administration of IL-10 depressed serum TNF-α production after HS in a rat model. A study by Lee, Lee (7) found that pretreatment with uvastatin resulted in a decrease in HS-induced serum TNF-α level and an increased IL-10 production. These results suggest that uvastatin reduced the production of proin ammatory cytokines and increases the production of anti-in ammatory cytokines, thereby preventing HSassociated multi-organ damage. Our study showed that serum TNF-α level tended to decrease in the HS + statin group compared to the HS group. However, pro-in ammatory cytokines were not signi cantly decreased in the HS + statin group. On the other hand, a study by Chen, Lee (5) revealed that uvastatin can increase plasma IL-10 production when given after the onset of sepsis in a rat model and may have therapeutic effects in treatment of LPS-induced shock. These results are consistent with our ndings. In this study, treatment with uvastatin after HS resulted in higher IL-10 production than in the HS-only group 2 hrs after HS. This result suggested that posttreatment with uvastatin elevates serum anti-in ammatory cytokines. However, these immunomodulatory effects were somewhat weak compared to pre-treatment with uvastatin. In addition, cytokine pro le differences had little effect on laboratory and other measured parameters in the HS + statin group. This may be associated with the therapeutic dosage or pharmacokinetics of uvastatin used in our study. A previous study on the effects of intravenous rosuvastatin for acute stroke showed that it provided protection at concentrations of 0.2 and 2.0 mg/kg, but not at 0.02 mg/kg,(27) which could have been a similar factor in our experiments. Moreover, because the elimination half-life averages 0.7 hours for intravenous uvastatin, multiple doses may be needed. (28) Further studies are required to investigate the optimal treatment dosage of uvastatin for post-treatment use in HS.
Our study had some limitations. First, we used a rat model of controlled HS, which does not completely re ect the clinical situation. However, because we wanted to compare the cytokine levels of four groups over time and needed to ensure similar blood loss between groups, a controlled model of HS was most appropriate. Moreover, the discrepancies in shock severity in an uncontrolled HS model could have acted as signi cant confounding factors. Second, the experiment was conducted with only 30% blood loss (moderate HS). Therefore, this amount of bleeding may have been insu cient to cause changes in the various cytokines that affect immune status in a rat HS model. Greater blood loss would likely lead to greater differences in the outcomes of uvastatin treatment for HS.

Conclusion
This study showed that post-treatment with uvastatin did not signi cantly suppress pro-in ammatory cytokines but did elicit a meaningful increase in anti-in ammatory cytokine IL-10 production. In this rat model, cytokine pro les after HS were altered by uvastatin treatment.  Data are presented as mean and S.D. Control group: did not receive drugs or undergo hemorrhagic shock (HS) procedures, control + statin group: received fluvastatin 1 mg/kg without HS, HS group: received normal saline after HS, HS + statin group: received fluvastatin 1 mg/kg + normal saline after HS. *P < 0.005 when compared with the control group and control + statin group ‡ P < 0.001 when compared with the control group and control + statin group Figure 1 Experimental protocol in hemorrhagic shock in rats  Change in mean arterial pressure (MAP) (a) and heart rate (HR) (b) in a hemorrhagic shock model. Data are presented as mean and S.E. * P < 0.05 for the HS group and HS + statin group compared with the control group and control + statin group. Control group: did not receive drugs or undergo hemorrhagic shock (HS) procedures, control