Ethics statement
All animal experiments were approved by the Ethics Committee of the Wuxi People’s Hospital (approval number: MS201916).
Animals
Thirty healthy male Sprague–Dawley rats (270–320 g) were acquired from Changzhou Cavens Laboratory Animal Co., Ltd. (Changzhou, Jiangsu, China) and housed in specific pathogen-free conditions with animal service from the Laboratory Center of Jiangsu Lung Transplantation in Wuxi. The room temperature was set at 23–25 °C with a 12-h day/night cycle, and the rats (n = 5 per cage) were acclimatized for 7 days with ad libitum access to water and food before the experiments [4]. The rats were fed and cared for by a full-time technician, who coded the cages and rats. The rats were randomly assigned to three groups using a computer-based random order generator (n = 10 each): (1) C group, in which the rats underwent abdominal and cervical incisions and sutures; (2) cecal ligation and puncture (CLP) group, in which the rats underwent CLP; and (3) CSTT group, in which the rats underwent isolation and transection of the sympathetic nerve trunk following CLP. For each animal, five different investigators were involved and had the following roles.The first investigator performed the procedures including sample collection based on the randomization table. This investigator was the only person aware of the treatment group allocation. The second investigator was responsible for performing blood gas analysis and serum biochemical assay, and the third investigator performed the western blot analysis and histopathological test. Finally, the fourth investigator (also unaware of treatment) assessed the animals’ behavior, and the fifth investigator performed data analysis.
Surgical procedures
The rats were anesthetized using 5% sevoflurane on an anesthesia machine for small animals followed by intraperitoneal injection of pentobarbital sodium (30 mg/kg) [5, 6]. All the rats in this trial maintained autonomous respiration and air inhalation during the whole procedure.They were maintained in a supine position on a heating table mat (AUX Group, Ningbo, Zhejiang, China) with the temperature set to 37 °C. Hair over the abdomen and around the neck was shaved using an electric haircutter. Anesthesia was verified by testing limb immobility via forceps clamping of the skin. The skin over the abdomen and neck was sterilized using 75% alcohol. Ophthalmic scissors were used for the incisions of the skin, abdominal muscles, and peritoneum along Hunter’s line of the lower abdomen at a width of 1.5–2.0 cm. An incision along Hunter’s line can prevent vascular injury and bleeding. The cecum, usually located in the right upper abdomen, was extracted using anatomical forceps. A 3–0 silk suture was passed through the mesocecum, and the cecum was ligated at its upper third adjacent to its root [7]. The cecum was then bilaterally punctured by inserting an 18-gauge needle through the cecal wall followed by cecal pressing with sterile cotton swabs and extrusion of the intestinal contents through the punctured openings. The cecum was returned to the abdominal cavity, and the peritoneal and skin incisions were sutured layer-by-layer using 3–0 absorbable sutures. Sham-operated mice in the C group underwent identical procedures except for CLP. All animals received an abdominal infusion of normal saline (1 mL) after closure of the abdominal wall.
The mice in the CSTT group underwent identical procedures for CLP with additional CSTT [8, 9]. The ventral neck was incised using ophthalmic microscissors to expose the platysma myoides and left sternocleidomastoid. The triangle of the left sternocleidomastoid was bluntly dissected using the ophthalmic microscissors until the carotid artery sheath was exposed. The carotid artery was isolated from the nerve trunk using ophthalmic microforceps. The top end of the forceps was arc-shaped with a diameter of 0.3 mm. With the vagus nerves isolated from the sympathetic nerve trunk using the ophthalmic microforceps, the sympathetic nerve trunk was transected using ophthalmic microscissors. The platysma myoides and skin were closed using 3–0 absorbable sutures. The index of a successful model establishment was the development of Horner’s syndrome, with features such as blepharoptosis, narrowing of the palpebral fissure, miosis, and canthus secretions. Conversely, the absence of Horner’s syndrome was considered an indication of a failed model, and another experiment was warranted. If the rats died during the procedure, another experiment was conducted until the desired number was achieved.
The rats were returned to their cages followed by care and ad libitum access to food and water in the laboratory for 24 h. The animals’ behavior; wet/dry lung weight ratio (W/D); oxygenation index (expressed as the ratio of arterial partial pressure of oxygen [PaO2] to oxygen concentration [FiO2]); lung injury scores assessed by histopathology analysis; serum concentrations of chemokines, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10; and sPLA2 levels were assessed.
Blood gas analysis
After 24 h, the rats were anesthetized, and the lower abdomen was opened through the original incision. The small intestine was extracted, and the mesentery was bluntly and gently isolated using sterilized cotton swabs. The abdominal aorta and inferior vena cava were then exposed. Blood from the aorta was obtained using a 1-mL syringe for arterial blood gas analysis. The oxygenation index was evaluated by dividing PaO2 by FiO2. We regarded FiO2 in the air as 21%. Venous blood was obtained from the inferior vena cava and stored in an anticoagulant-coated tube. Venous blood was centrifuged, and the serum was stored in a refrigerator for subsequent analysis.
Histopathological analysis
The rats were euthanized by cervical dislocation. The pulmonary artery was irrigated with normal saline until the absence of blood was noted from the lungs. With the superior lobe of the right lung isolated, water and blood were removed using a filter paper, and the wet weight was measured. Then, the sample was kept in a drying oven at 120 °C for 24 h; subsequently, the dry weight was estimated, and the W/D was calculated. The middle lobe of the right lung was dissected, fixed with 10% formaldehyde solution, embedded in paraffin, microtomed into slices, and stained with hematoxylin–eosin. The slides were observed under a light microscope (100 × and 200 × magnifications). Histological changes were evaluated by a pathologist blinded to the experimental conditions. The degree of lung injury was graded using a histological scoring system [10]. Edema, alveolar and interstitial inflammation and hemorrhage, atelectasis, necrosis, and hyaline membrane formation were scored on a 5-point scale as follows: 0, no injury; 1, injury in 25% of the viewing field; 2, injury in 50% of the viewing field; 3, injury in 75% of the viewing field; and 4, injury throughout the field. Three different viewing fields per slide were analyzed, and the mean score of the three viewing fields was considered the score of the slide. The final lung injury score was obtained by summing these scores [11].
Serum biochemical indices
The serum concentrations of chemokines, TNF-α, IL-6, and IL-10 were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Neobioscience, Nanjing Proteinbio Technology Co., Ltd., China) according to the manufacturer’s instructions [12]. The plates were read using a microplate reader at a wavelength of 450 nm [13].
Western blotting
sPLA2 levels were determined using western blotting according to the instructions of the Protein Assay Kit (Abcam, Cambridge, Univ Biotechnology Co., Ltd., Shanghai, China, ab139692) [14]. The left lung was dissected; 100 mg was cut into pieces before being placed into the centrifuge tube, and 1 mL of lysis solution was injected into the tube. A total of 50 μL of lysis solution included 40 μL of radioimmunoprecipitation assay, 5 μL of the protease inhibitor, 5 μL of the phosphatase inhibitor, and 0.5 μL of phenylmethanesulfonyl fluoride. Two 50-mL beakers were prepared. One beaker was filled with normal saline as a wash homogenizer, and another one was filled with ice to keep the centrifuge tube filled with lung tissue cool. Homogenization was performed for 2 s. The centrifuge was placed in a refrigerator at 4 °C for more than an hour before being used. The lysed tissue was centrifuged for 10 min at 15,000 rpm and 4 °C after being lysed for 30 min at 4 °C. About 800 μL of supernatants were obtained.Samples from the cell supernatants were subjected to 15% lauryl sodium sulfate–polyacrylamide gel electrophoresis followed by immunoblotting onto a polyvinylidene difluoride membrane. Nonspecific binding sites were blocked via incubation with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 4 h at 4 °C. The membranes were then incubated overnight. After rinsing with TBS-T, the blots were incubated for 2 h at room temperature. The immunoreactive bands were visualized using an enhanced chemiluminescence detection kit, and the intensities were analyzed using an image processing program. Equal protein loading was confirmed using horseradish peroxidase-conjugated anti-mouse IgG (ab205719; Abcam, Cambridge, UK) as the secondary antibody.
Statistical analysis
Values are expressed as mean ± standard deviation of three experiments. Comparisons were assessed using repeated measurement variables (within-subjects factors) followed by an independent samples t-test. Data were tested using the D’Agostino–Pearson test for normal distribution. Data were analyzed using MedCalc (version 20.110–32-bit;MedCalc Software Ltd., Ostend, Belgium). The level of statistical significance was set at p < 0.05.
