Ropivacaine

All patients agreed to receive subarachnoid anesthesia [Ropivacaine (AstraZeneca AB, 20–25 mg)] by an experienced anesthesiologist. This was initiated immediately following CD. PCIA was administered using an electronic infusion pump containing 800 mg tramadol (Sandoz Pharmaceutical Co. Ltd., China) in 0.9% normal saline (NS, 300 ml). The background volume was 6.0 ml/h, and the patient-controlled analgesia (PCA) dose was 2 ml, with a locking time of 15 min. Acetaminophen and NSAIDs were not administered, and tramadol was the only rescue analgesic for inadequate analgesia.

Right tibial fracture with intramedullary fixation procedure was performed under anesthesia to establish the POCD animal model described in the previous study (Chen et al., 2019 (link)). Mice were subjected to 3% isoflurane for induction and 1.5% isoflurane for maintenance. Then an incision was made lateral to the right tibia to expose the bone and then a 0.38 mm intramedullary fixation needle was inserted into the tibial medullary canal for fixation. Finally, an osteotomy was performed in the middle and distal thirds of the tibia. The surgical incision was sutured with 4–0 silk non-absorbable suture (Mersilk; Ethicon, United States). The mice body temperature was adjusted at 36–37^°C during the whole procedure by a heating pad. Ropivacaine (0.2%, Oxford; AstraZeneca) was applied locally to prevent postoperative pain.

Control group: The cocktail was prepared into a 100 ml solution consisting of 200 mg of ropivacaine injection (Astra Zeneca AB, Import Drug Registration Certificate H20140763) + 5mg of morphine hydrochloride injection (Northeast Pharmaceutical Group Company Shenyang No.1 Pharmaceutical Co., Ltd., National Drug Approval No. H21022436) + normal saline. 50ml of the above cocktails were extracted and added into 40mg of triamcinolone acetonide injection (Kunming Jida Pharmaceutical Co., Ltd., State Drug Approval No. H53021604). 50ml of the above cocktail was drawn and added 40mg of triamcinolone acetonide injection (Kunming Jida Pharmaceutical Co., Ltd., National Medicine Standard H53021604), with the injection site at the posterior joint capsule; 50ml cocktail without hormones was injected into MCL, LCL, quadriceps, subpatellar fat pad, other joint capsules and subcutaneous locations. Experimental group: 50mg flurbiprofen axetil injection (Beijing Tide Pharmaceutical Co., Ltd., State Drug Approval No. H20041508) was added into the above cocktails, and the other drug use and administration methods were the same as those in the control group. Remedial analgesia regimen: Patients were given dezocine (Yangtze River Pharmaceutical Group Co., Ltd., State Drug Approval No. H20080329) intramuscular injection in case of pain visual analogue scale (VAS) ≥ 4 points.

In preparation for surgery, the neck area was cleaned with alcohol and betadine alternately after the mice were anesthetized. An incision of approximately 5 mm was made on the left side of the throat, 2–3 mm from the clavicle. In order to visualize the carotid jugular bundle, the fatty tissue and fascia were gently separated from salivary glands. A small incision was made to expose the vagus nerve between the carotid artery and the jugular vein. For anesthetization, ~39.1 mg/kg ropivacaine (AstraZeneca) was injected into the vagus nerve.^{48 (link)} For resection, the vagus nerve was firmly grasped and pulled toward the head of the mouse until the vagus broke.

A nurse, who will open the sealed envelopes, will prepare the syringe containing either 0.3 ml/kg of 0.33 % ropivacaine (AstraZeneca Pharmaceutical, Inc., London, UK) or 0.3 ml/kg of saline for DPNB, and 10 ml of 1 % tetracaine gel (Xi’an Lijun Pharmaceutical Co., Ltd., Xi’an, China) or 10 ml of liquid glycerine (YunJia Medical Technology Co., Ltd., Harbin, China) for instillation. The participants, attending anesthesiologist and urologist, as well as the researchers will be not aware of the randomization. The allocation concealment will not be exposed until the final data analysis report is completed.

Human lung cancer cell lines (A549 and H1299), which are two of the common lung cancer phenotypes clinically, were purchased from the Cellular Biology Institute of the Shanghai Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 medium (GIBCO, United States) supplemented with 10% bovine serum (Biological Industries, Beit HaEmek, Israel). The cells were grown in monolayer at 37°C in a humidified atmosphere supplemented with 5% CO₂. Ropivacaine (AstraZeneca AB, Sweden) was dissolved in normal saline, with the pH adjusted to 7.4, and kept at −20°C. The cultured lung cancer H1299 and A549 cells at 90% confluence were treated with Ropivacaine at 0.5, 1, and 2 mM. Cells treated with saline served as controls. Cobalt chloride (CoCl₂) (Sigma-Aldrich, St Louis, MO, United States) at 100 μM was used to induce cellular hypoxia and increase HIF-1α expression in both A549 and H1299 cells treated with Ropivacaine.