Thirty-two male Wistar-Kyoto rats weighing 260–300 grams were purchased from the National Animal Center (Taipei, Taiwan). They were housed in the university Animal Center in a controlled environment at a temperature of 22±1°C with a 12-h light/dark cycle. Food and water were provided ad libitum. The Animal Care and Use Committee of Tzu Chi University approved the experimental protocol. The animals were anesthetized with ether inhalation for about 15 min. During the period of anaesthesia, a polyethylene catheter (PE-50) was inserted into the femoral artery to collect blood samples and was connected to a pressure transducer (Gould Instruments, Cleveland, OH, USA) to record arterial pressure (AP) and heart rate (HR) on a polygraph recorder (Power Lab, AD Instruments, Mountain View, CA, USA). Another PE-50 catheter was inserted into the femoral vein for intravenous administration of drugs or fluid. The operation was completed within 15 min, leaving a small wound (less than 0.5 cm²). After the operation, the animals were placed in a conscious rat metabolic cage (Shingshieying Instruments, Hualien, Taiwan). Rats awakened soon after the operation and HS was induced 24 h later, with the rats in a conscious state [⁹–¹¹].

HS was induced by drawing blood from the femoral arterial catheter into a 10 ml syringe. An infusion pump controlled the withdrawal rate to mimic a typical bleeding event. The blood volume withdrawn was 60% of the total blood volume (6 ml/100 gm BW). The duration of blood withdrawal was 30 min. Within these 30 min blood was withdrawn and the HS-procedure was carried out, followed by resuscitation with 0.5 ml normal saline at 0, 1, 3, 6, 9, and 12 h after HS. After blood withdrawal, the animals were continuously observed for 48 h and sacrificed later for pathology [⁹–¹¹].

The animals were randomly divided into 4 groups. Rats in the Rosiglitazone group (n=8), were not subjected to HS and were given 0.3 mg/kg rosiglitazone (GlaxoSmithKline Taiwan Ltd, Taipei, Taiwan) in 0.5 ml normal saline intravenously over over a period of 10 min [¹²]. Rats in the HS group (n=8), HS was induced and the rats immediately received an intravenous drip of 0.5 ml normal saline over over a period of 10 min. In the Rosiglitazone + HS group (n=8), HS was induced and the rats immediately received 0.3 mg/kg rosiglitazone intravenously over over a period of 10 min. In the Control group (n=8), rats were not subjected to HS and immediately received an intravenous drip of 0.5 ml normal saline over over a period of 10 min.

Arterial blood samples were obtained for baseline values before heparinization. Heparin (2 IU/gm BW) in 1 ml normal saline was injected via the catheter into rats over a period of 20 min [⁷–⁹]. Arterial blood samples (0.5 ml) were collected for measurement of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), creatine phosphokinase (CPK), lactate, and glucose at 30 min before induction of HS and at 0, 1, 3, 6, 9, and 12 h after HS while an equal volume of 0.5 ml normal saline was used. Blood samples were immediately centrifuged at 3000 g for 10 min. The serum was decanted and separated into 2 parts; 1 part was stored at 4°C within 1 h after collection for biochemical analysis. Serum levels of GOT, GPT, BUN, Cre, LDH, CPK, lactate, and glucose were measured with an autoanalyzer (COBAS Integra C111, Roche Diagnostics, Basel, Switzerland) to obtain various biochemical data. The other part of the serum collected at 1, and 12 h after HS was stored at −80°C for later measurement of TNF-α, IL-6, and MCP-1 concentrations [⁹–¹¹].

After 1 h and 12 h of the induction of HS, TNF-α, IL-6, and MCP-1 concentrations in the blood samples were measured separately by antibody enzyme-linked immunosorbent assay (ELISA) with commercial antibody pairs, recombinant standards, and a biotin-streptavidin-peroxidase detection system (Endogen, Rockford, IL, USA) as described previously [⁹–¹¹]. Blood samples were collected in serum-separated tubes. All reagents, samples, and working standards were brought to room temperature and prepared according to the manufacturer’s directions. Reactions were quantified by optical density using an automated ELISA reader (Sunrise, Tecan Co., Grödingen, Austria) at 450/540 nm wavelength.

The liver, kidney, lung and small intestine were removed immediately after sacrifice at 48 h after induction of HS. Examples of the particular type of these tissues specimens were fixed overnight in 4% buffered formaldehyde, processed using standard methods, and stained for hematoxylin and eosin (H & E). One observer, who was blinded to the group assignment, performed the tissue analysis. The severity of liver injury observed in the tissue sections was scored as follows: 0, no evidence or minimal evidence of injury; 1, mild injury consisting of cytoplasmic vacuolation and focal nuclear pyknosis; 2, moderate to severe injury with extensive nuclear pyknosis, cytoplasmic hypereosinophilia, and loss of intercellular borders; and 3, severe necrosis with disintegration of the hepatic cords, hemorrhage, and neutrophil infiltration [⁹,¹⁰]. The severity of renal tubular injury was scored by estimating the percentage of tubules in the cortex or the outer medulla that showed epithelial necrosis or had luminal necrotic debris, tubular dilation, and hemorrhage: 0, none; 1, <5%; 2, 5% to 25%; 3, 25% to 75%; and 4, >75% [⁹–¹¹]. Lung injury was scored as follows: 0, no evidence; 1, mild injury; 2, moderate injury; 3, severe injury with lung oedema, interstitial inflammatory cell infiltration, and hemorrhage [⁹–¹¹]. The severity of small intestine injury was scored from 0 to 3 as follows: 0, normal (no damage); 1, mild (focal epithelial edema and necrosis); 2, moderate (diffuse swelling or necrosis of the villi); 3, severe (diffuse necrosis of the villi with evidence of neutrophil infiltration in the submucosa and/or hemorrhage) [⁹–¹¹]. All evaluations were made on 5 fields per section and 5 sections per organ.

Data are expressed as mean ±SD. Statistical comparisons between different groups at corresponding time points were made by repeated measures of 2-way ANOVA followed by a post hoc test (Bonferroni’s method). Survival analysis was performed using the Kaplan-Meier estimator and comparisons among the mortality rates of the groups were made with the log rank test. Histological scores were analyzed by the Kruskal-Wallis test followed by the Dunn’s test. A p value less than 0.05 was considered statistically significant.

All rats were alive at 12 h after induction of HS. However, in the Rosiglitazone + HS group the rats were dead by 18 h after induction of HS, and in the HS group the rats were dead by 24 h after induction of HS. The survival rate at 48 h after induction of HS was 50% in the HS group, 100% in the Control group, 100% in the Rosiglitazone group, and 75% in the Rosiglitazone + HS group (Figure 1A). The mortality rate between groups in the Rosiglitazone + HS group was significantly lower than in the HS groups (log rank test; p=0.021). Mean arterial pressure (MAP) decreased rapidly after withdrawal of 60% of total blood volume from the femoral arterial catheter in rats. MAP stayed relatively low during 12 h after induction of HS (Figure 1B). Compared with the HS group, treatment with rosiglitazone did not affect the MAP after HS (Figure 1B). The heart rate (HR) was significantly increased during HS (Figure 1C). Compared with the HS group, HR in the rosiglitazone + HS group did not change after HS (Figure 1C).

GOT was gradually increased after induction of HS (Figure 2A). Compared with the HS group, treatment with rosiglitazone decreased GOT at 3, 6, 9, and 12 h after induction of HS (^#P<0.05; Figure 2A). Serum GPT increased at 0, 1, 3, 6, 9, and 12 h after HS compared with the Control group (*P < 0.05; Figure 2B). Compared with the HS group, treatment with rosiglitazone decreased the GPT at 3, 6, 9, and 12 h (^#P<0.05; Figure 2B). The GOT and GPT were no statistically different between the Rosiglitazone group and the Control group.

HS increased blood BUN to a peak at 6 h (Figure 2C). Compared with the HS group, treatment with rosiglitazone decreased the BUN at 1, 3, 6, 9, and 12 h (^#P<0.05; Figure 2C). Serum Cre increased rapidly after induction of HS. The values of serum Cre increased at 0, 1, 3, 6, 9, and 12 h after HS, compared with the Control group (*P<0.05; Figure 2D). Compared with the HS group, treatment with rosiglitazone decreased the serum Cre at 0, 1, 3, 6, 9, and 12 h after HS (^#P<0.05; Figure 2D). The BUN and Cre were no statistically different between the Rosiglitazone group and the Control group.

LDH was gradually increased after induction of HS (Figure 3A). Compared with the HS group, treatment with rosiglitazone decreased the LDH at 0, 1, 3, 6, 9, and 12 h after induction of HS (^#P<0.05; Figure 3A). Blood CPK increased gradually after induction of HS (Figure 3B). Compared with the HS group, treatment with rosiglitazone decreased the CPK at 6, 9, and 12 h after induction of HS (^#P<0.05; Figure 3B). The LDH and CPK were no statistically different between the Rosiglitazone group and the Control group.

Serum lactate increased at 0, 1, 3, 6, 9, and 12 h after HS, compared with the Control group (*P<0.05; Figure 3C). Compared with the HS group, treatment with rosiglitazone decreased the serum lactate at 1, 3, 6, 9, and 12 h after HS (^#P<0.05; Figure 3C). Blood glucose increased to a peak at 3 hr (Figure 3D) and returned to baseline at 12 hr after induction of HS. Compared with the HS group, the Rosiglitazone + HS group demonstrated decreased glucose at 0, 1, 3, 6 and 9 h (^#P<0.05; Figure 3D). The lactate and glucose were not statistically different between the Rosiglitazone group and the Control group.

HS greatly elevated the serum TNF-α compared with the Control group at 1 and 12 h after induction of HS (*P<0.05; Figure 4A). Treatment of rosiglitazone significantly decreased the serum TNF-α at 1 and 12 h after induction of HS (^#P<0.05; Figure 4A). HS increased the serum IL-6, compared with the Control group at 1 and 12 h after induction of HS (*P<0.05; Figure 4B). Rosiglitazone decreased the serum IL-6, compared with the HS group at 1 and 12 h after induction of HS (^#P<0.05; Figure 4B). HS increased the serum MCP-1 compared with the Control group (*P<0.05; Figure 4C). Treatment with rosiglitazone decreased the serum MCP-1 compared with the HS group after induction of HS (^#P<0.05; Figure 4C). The TNF-α, IL-6 and MCP-1 were not statistically different between the Rosiglitazone group and the Control group.

After the induction of HS at 48 h, only 4 rats were alive in the HS group, 6 rats were alive in the Rosiglitazone + HS group, and all rats were alive in the Control group. These rats were sacrificed and arranged for pathology study. H & E-stained tissue sections from the liver (Figure 5I), kidney (Figure 5J), lung (Figure 5K), and small intestine (Figure 5L) of the Control group, and the liver (Figure 5M), kidney (Figure 5N), lung (Figure 5O), and small intestine (Figure 5P) of the Rosiglitazone group are shown in Figure 5. Compared with the Control group, histopathologic analysis of H & E-stained tissue sections from the liver, kidney, lung, and small intestine after HS revealed hepatic cells necrosis and hemorrhage in the liver (Figure 5A), tubular cells swelling, nuclear loss, tubular dilatation, brush border loss and hemorrhage in the kidney (Figure 5B), pulmonary edema, hemorrhage and interstitial cell infiltration in the lung (Figure 5C), and loss of epithelial and mucosal architecture, and destruction of the vili in the small intestine (Figure 5D). Compared with the HS group, treatment with rosiglitazone abolished the histopathologic changes in the liver, kidney, lung and small intestine (Figure 5E–H). Compared with the HS group, the injury scores of the liver, kidney, lung, and small intestine were significantly decreased in the Rosiglitazone + HS group (^#P<0.05; Figure 5Q–T).