Methods
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 fluid infusion and intravenous drug administration. The surgical procedure was completed in 15 minutes, and the incision was less than 0.5 cm2 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 fluvastatin (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 fluvastatin + normal saline after HS induction. Fluvastatins were given immediately after the first 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 significant 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 Blood was withdrawn through the femoral artery until MAP decreased to 60 mmHg.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 fluid 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. Arterial blood gas testing was conducted using a point-of-care laboratory instrument (iSTAT, Abbott, Abbott Park, IL, USA) and test cartridges (CG3; Abbott). CBC testing was conducted using a CBC measuring instrument (Hemavet 950FS, Drew Scientific, USA). Serum cytokines including interleukin (IL)-1β, IL-6, IL-10, IL-12p70, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were measured at baseline, 2 hrs after administration of shock, and 48 hrs after resuscitation. IL-1β, IL-6, IL-10, IL-12p70, IFN-γ, and TNF-α concentrations in blood samples were measured using a Multiplex kit (Bio-Rad, San Diego, USA) and run on Luminex technology (Bio-Plex Multiplex Bead Array System, Bio-Rad Hercules, CA, USA) according to the manufacturer’s instructions.
Histological examination
Lung, duodenum, and kidney specimens were removed immediately after sacrifice, fixed 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 five fields per section and five sections of lung, duodenum, and kidney.