Animal preparation
Twenty-one healthy domestic male Beijing Landrace pigs (3 months, 30 ± 2 kg) were fasted overnight with free access to water. The animals were initially sedated with a single intramuscular injection of midazolam (0.5 mg/kg). Anesthesia was induced with a bolus dose of propofol (1 mg/kg) and maintained with pentobarbital (8 mg/kg/h) by ear vein. A cuffed 6.5 mm endotracheal tube was advanced into the trachea. Animals were mechanically ventilated with a volume controlled ventilator (Evita4, Drager Medical, Lubeck, Germany) using a tidal volume of 10 ml/kg and FiO2 at 0.21. End-tidal PCO2 was continuously monitored by in-line infrared capnography to maintain at 35–45 mmHg by adjusting respiratory frequency. A vesical catheter with a thermometric detector was intubated into the bladder to measure body temperature and monitor urine output. The left femoral artery was dissected to insert a 4-F arterial catheter into the descending aorta to measure arterial pressure (MAP). A 6-F arterial sheath was inserted into the right femoral artery for rapid bleeding. A 5-F central venous catheter was inserted via the left femoral vein for volume resuscitation. Room temperature was adjusted to 26 ℃. Body temperature was maintained above 37 ℃ with a heating pad.
Experimental protocol
After operation, the animals were allowed to stabilize for 30 min, and baseline data were obtained. HS was induced by rapid bleeding via the arterial sheath to a MAP of 40 mmHg within 10 minutes and maintained at 40 ± 3 mmHg for 60 minutes [12]. Shed blood was collected in sterile bags (S-400, Sichuang Nightingale Biological Co. Ltd). Additional blood was withdrawn at a MAP ≥ 44 mmHg, or crystalloid solution was infused at a MAP ≤ 36 mmHg. Volume resuscitation started after 1 hour of shock. The animals were resuscitated with the shed blood during the first hour of reperfusion, and then received a basal crystalloid infusion of 10 ml/kg/hr. At 6 hours after the onset of HS, animals were placed in a lateral position. A 10 cm mid-sagittal incision was made over the frontal and parietal portions of the scalp. A 15 mm parietal cranial window was opened by drilling. Then dura mater was incised to observe the cerebral microcirculation as our previous experiment [13]. Animals were euthanized with an intravenous injection of 100 mg/kg pentobarbital after recording microcirculation, and then cortical tissue samples from parietal lobe were removed. Some samples were frozen in liquid nitrogen and stored at −80 °C for protein analysis. Some samples were stored in 4% paraformaldehyde.
Five pigs were randomly assigned to the sham operation (SO) group, which had no HS or volume resuscitation. Sixteen pigs of HS were randomly divided into two groups: (1) SFI group (SFI, n=8): conventional volume resuscitation and SFI at a dose of 3 ml/kg; (2) saline group (SA, n=8): conventional volume resuscitation and saline at a dose of 3 ml/kg.
Measurements
Microcirculatory imaging
Cerebral microcirculation was recorded by a SDF imaging video microscope (MicroScan; MicroVision Medical Inc, Amsterdam, The Netherlands). Individual videos of 10 seconds were analyzed off-line using a score, in which 0 represents no flow, 1 represents markedly reduced flow, 2 represents reduced flow, and 3 represents normal flow. The microvascular flow index (MFI) score represented the average value of the predominant type of blood flow. Following the guidelines [14], we determined MFI both for large microvessel (diameter > 20 µm) and small microvessel (diameter < 20 µm). To avoid possible bias, the analysis of the videos of the microcirculation was blinded.
Immunohistochemistry (IHC)
IHC was used to evaluate the protein expression of NOS in brain tissues. Cortical tissues fixed with 4% paraformaldehyde were dehydrated with alcohol solutions of gradient concentration (100, 95, 80 and 75%), sliced at 4 μm thickness, and embedded in paraffin. The sections were placed in histosol for the removal of paraffin, rehydrated in graded ethanol, and blocked in 5% bovine serum albumin (Sigma‑Aldrich; Merck KGaA, Darmstadt, USA) for 4 hours. Sections were incubated with eNOS antibody (1:1000 dilution; rabbit. no. Ab5589, Abcam Co, China), iNOS antibody (1:1000 dilution; rabbit. no. Ab1956, Abcam Co, China), and nNOS antibody (1:1000 dilution; rabbit. no. Ab3342, Abcam Co, China) at 4 ˚C overnight. The sections were then incubated with biotinylated goat anti-rabbit secondary antiserum. After counterstaining with hematoxylin, Image J software (National Institutes of Health, Bethesda, MD, USA) was used to determine the optical density of the images. Five sections were selected from each group, and the average values were measured.
IL 6 and TNF-α in the brain tissue
Brain homogenates were obtained from the cortical tissue and centrifuged at 2000 x g for 15 minutes to remove cellular debris. The levels of IL 6 and TNF-α were measured using specific enzyme-linked immunosorbent assay kits according to the manufacturer's instructions.
Western blotting
AQP 4 protein expression in cortical tissue was measured by western blotting according to manufacturer’s instruction. Integrated optical densities of protein bands were digitally quantified using Gel-Pro Analyzer version 3.1 (Media Cybernetics, Silver Spring, MD). Protein levels were normalized to β-actin and presented as a ratio.
Brain ultrastructure
Tissue samples of the cerebral cortex (preserved in 4% paraformaldehyde) were used to assess ultrastructural changes of microvascular endothelia under a transmission electron microscope (H-7650; Hitachi, Tokyo, Japan).
Statistical analysis
Data of normal distribution were expressed as mean ± SD. One-way analysis of variance was used to assess the differences among the three groups followed by Bonferroni’s post hoc test to correct for multiple comparisons. Student's t-test was used for the comparisons of shed blood between SFI and SA groups. A p-value < 0.05 was considered significant. Statistical analysis was performed with SPSS 19.0 software (SPSS Inc, Chicago, IL) and GraphPad Prism version 5 (GraphPad Software, San Diego, CA).