Microsurgical, surgical microscopy and conventional surgical instruments were purchased from the Carl Zeiss Company (Germany). A respiratory anesthesia machine and a small animal ventilator were purchased from Surgivet and Harvard Apparatus (USA). The Powerlab multichannel physiological monitor was a product of AD Instruments Corporation (USA). The blood gas analyzer was a product of the NOVA Biomedical company (USA). A stocker double head roller pump was purchased from Stockert company (Germany). The variable temperature blanket was from ATC1000 (Sarasota, USA). The Jostra HCU20-600 variable temperature water tank was from the Maquet Critical Care AB, Solna, Sweden. Special small animal lung membranes were produced by Dongguan Kewei Medical Supplies Ltd. (China). The thermostat was purchased from Xijing Medical Supplies Co., Ltd. (China). The anesthesia trocar (16G, 20G, 22G) was from United States Becton Dickinson company. Venous drainage tube syringes (1 ml, 2 ml, 5 ml, 20 ml) were obtained from Beijing Freseniuskabi Pharmaceutical Co., Ltd. (China). The surgical suture was from Johnson & Johnson (USA). The glass thermometer and the Flow cytometry Model LSR II were from BD (Biosciences; USA). Desktop Centrifuge was purchased from Beckman Coulter (USA). The electronic balance was purchased from the Beijing Sartorius company. The − 80 °C and − 20 °C refrigerants were obtained from the Germany SIEMENS company. The thermostatic temperature water bath was from Shanghai Yuejin Medical Instrument Factory (China). The biological tissue paraffin embedding machine and Stapler were purchased from the Leica company (Germany). The single-channel micropipettes were from Gilson (Germany). The spectrophotometer was from Shanghai Tianpu Analytical Instrument Co., Ltd. (China). The microplate reader and SoftMax Pro microplate reading software SpectraMaxPlus384 were from BD, USA. The swirling Mixer was from Lin Bell Instrument Manufacturing Co., Ltd. (China). The BX51 optical microscope and DP71 camera system were from Olympus Corporation (Japan). The JSM-5500 scanning electron microscopy was from JEOL Co., Ltd. (Japan). The Hitachi Model H-7650 transmission electron microscope was from Hitachi (Japan). The TEM imaging system was from AMT imaging (MA, USA). The glass slide for fixed tissues was from Wuhan Boster Biological Technology Co. (China). The EP tube, tissue homogenizer, frozen tube, biochemical blood tubes, and test tube liquid nitrogen biological containers were from Wuhan Boster Biological Technology Co. (China).
6% HES130/0.4 was purchased from Beijing Fresenius Kabi Pharmaceutical Co., Ltd. (China). Heparin was obtained from Changzhou Qianhong Biochemical Pharmaceutical Co., Ltd. (China). The myeloperoxidase (MPO), superoxide dismutase (SOD), and malondialdehyde (MDA) assay kits were from the Nanjing Jiancheng Research Institute of Bioengineering (China). The BD CBA Mouse/Rat Soluble Protein Master Buffer Kit, Rat IL-10 Flex Set (A8), IL-1a Flex Set (A4), IL-4 Flex Set (B9), IFN-y Flex Set (A6), TNF Flex Set (C8) and the BD CBA Mouse/Rat Soluble Protein Master Buffer Kit were purchased from BD (USA).
Thirty healthy adult Sprague‒Dawley rats weighing approximately 350–450 g were provided by SLAC Laboratory Animal Company (Changsha, China) and kept with clean grade and feeding conditions according to the SPF standard. Before the experiment, rats received 12 h of fluorescence/12 h of dark room temperature in the animal feeding room in the Central South University Animal Center. The experimental study was approved by the Institutional Animal Care and Use Committee (IACUC) of The Second Xiangya Hospital, Central South University. All rats were sacrificed by cervical dislocation to collect intestinal tissue for follow-up experiments.
The rats were fasted for 12 h with free access to water before surgery. The rats were randomly selected and weighed. Before the operation, the time point T1 was recorded. For surgical procedures, the rats were first inducted for inhalation anesthesia with sevoflurane and then restrained. To monitor the rectal temperature, the thermometer lubricated by paraffin oil was inserted into the anal cavity. The rats were anesthetized with 100% pure oxygen mixed with 3% sevoflurane using a 16 G anesthesia cannula. The initial respiratory frequency was set at 60 beats/min, and the tidal volume was 8–10 ml/kg, which can be adjusted according to the blood gas value. After the operation, the rats were anesthetized by mechanical ventilation. Three vascular puncture tubes were punctured into the tail artery, left femoral artery, and right jugular vein under the surgical microscope operation. The left femoral artery was used for continuous hemodynamic monitoring during the bypass procedure; the caudal artery was used to connect the arterial end of the extracorporeal circuit, and the artery was perfused; the right jugular vein was connected to the venous drainage tube to the venous end of the extracorporeal circuit. The venous blood of the rats was drained. The left femoral artery was punctured with a 22 G trocar and 400 IU/kg heparin to heparinize the rat; the caudal artery was punctured with a 20 G cannula and connected to the perfusion end of the extracorporeal circulation; intravenous catheterization of the right jugular vein was performed using a modified 22 G IV silicone tube. Fine surgical scissors were used to cut the jugular vein. Under the guidance of the arterial sheath guidewire, the right jugular vein was drained into the right jugular vein. The distance was determined and labeled in the experiment), and the gravity height of venous drainage was 30–35 cm. All the distal vessels of the vessels were ligated with silk thread, and the proximal cardiac thread was fixed to prevent slipping. After the extracorporeal circulation pipeline connection, the crystal liquid was precharged with exhaust gas, and colloidal liquid (6% HES130/0.4) was used to replace the crystal liquid, keeping the liquid level at 1–2 ml. This model is a bloodless prefilled model. After catheterization, the venous drainage tube was placed in the reflux chamber, and the pliers were opened to start the cardiopulmonary bypass. To maintain the backroom fluid surface balance, the main pump diversion speed was adjusted. A membrane lung oxygen-air mixture of oxygen was used to maintain the flow of gas during the bypass of PaCO2 and PaO2 in the normal range. When the blood pressure dropped during the bypass, the liquid level could not be maintained, so more liquid was added to keep the blood pressure and fluid colloid level stable. There were no vasoactive drugs used during the entire experimental process. The temperature of the rats was lowered to the target temperature by using a variable temperature water tank, and the expected cooling time was 30 min.
Stopping cycle: cooling to the target temperature before the preparation of the prestop recording time T2; after stopping the venous end to continue drainage, take 20 ml syringe from the reflux chamber out of rat blood storage until the blood pressure reaches a straight line and the rat heart stops beating only. The cycle time was maintained for 30 min, and the cycle was stopped if the body temperature increased. The surface of the body evenly crushed ice to maintain the temperature.
Rewarming stage: at the beginning of cardiopulmonary bypass, the syringe was slowly reinjected into the blood chamber. Then, the temperature to rectal temperature was increased to 35 degrees Celsius, the rewarming time was expected to be 30 min, the temperature to the rectal temperature was approximately 35 degrees Celsius, and the rewarming time was 30 min. However, the rewarming time was kept as long as possible within one hour according to the actual situation. When the heart resumes, the time point T3 is recorded.
Stopping stage: After the temperature reached the target temperature, the machine was stopped, and the time point T4 was recorded before stopping. 5. After the operation, the ventilator was maintained for 60 min after cardiopulmonary bypass, and the time point T5 was recorded.
A total of 3 ml of whole blood from rats was extracted at the T1 time point, and 5 ml of whole blood from rats were extracted at the T5 time point. Whole blood was collected, placed in a 5 ml centrifuge tube, and placed on ice. After centrifuging at 3000 rpm for 15 min, the upper serum was kept at − 80 °C until use.
The intestine samples were cut to a 5 mm × 5 mm × 5 mm size and fixed into 10% formalin fixative (phosphate buffer pH 7.0) for hematoxylin–eosin (HE) staining. The intestine samples were quickly put into the precooled balanced salt solution; a 2 mm × 2 mm × 2 mm tissue sample was placed in 4% glutaraldehyde fixed for electron microscopy scanning or fixed with 1% osmium tetroxide for scanning electron microscopy scanning. The remaining parts were packed in cryopreserved tubes, frozen in liquid nitrogen, and stored at − 80 °C for further experiments.
Serum biochemical marker analysis
The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), blood urea nitrogen (BUN), and lactate dehydrogenase (LDH) were measured by an automatic biochemical analyzer.
Transmission electron microscope and scanning electron microscope analysis
For transmission electron microscope analysis, the fixed tissue samples were embedded in Spurr resin and sliced using an ultramicrotome (LEICA EM UC7). The samples were then stained with uranyl acetate and alkaline lead citrate (each for 5 to 10 min) and observed in a Hitachi Model H-7650 TEM with AMT TEM imaging system. For scanning electron microscope analysis, the fixed tissue samples were dehydrated and dried, covered with gold and examined under JSM-5500 scanning electron microscope.
The fixed specimens were paraffin-embedded and sliced to a thickness of 5 μm. Then, the slices were stained with hematoxylin and eosin, dehydrated with ethanol, cleared with xylene, sealed with neutral gum, and covered with a coverslip for the optical microscope observation.
Tissue SOD, MPO, and MDA activity detection
The tissue homogenate diluted with saline buffer was centrifuged at 2500 r/min for 10 min, and then the subsequent activity detection assay was carried out. The enzyme activity was determined according to the ELISA kit. The SOD activity unit is regarded as 50% inhibition of SOD per mg of tissue protein in a 1 ml reaction. For MPO, the enzyme activity unit was calculated as the decomposition of 1 mmol of H2O2 per gram of wet tissue at 37 °C in the reaction system.
The cytokines from each sample were detected using a Cytometric Bead Array (BD) and analyzed using a BD LSR II flow cytometer. The standard operation was carried out according to the instructions.
Experimental data acquisition
The blood pressure and heart rate of the rats were monitored continuously by LabChart7.0 software (AD Instruments Co., Ltd.); the rats were infused with 1 ml of heparin sodium salt water at the five time points T1, T2, T3, T4 and T5. Arterial blood samples were taken from the rats for blood gas analysis.
Statistical analysis was performed using GraphPad Prism 5.0 software. All data were analyzed by one-way ANOVA/two-way ANOVA followed by post hoc Tukey's test. P < 0.05 was considered to be statistically significant.