U2500

All animals in the study were anaesthetized with urethane (U2500; Sigma, St. Louis, MO, USA). The beta adrenoceptor agonist clenbuterol hydrochloride (clen; C5423; Sigma, St. Louis, MO, USA) and the beta adrenoceptor antagonist propranolol hydrochloride (prop; P0084; Sigma) were administered to the rats. Next, the muscarinic receptor antagonist atropine hydrochloride (atropine; A6625; Sigma) and the agonist acetylcholine hydrochloride (ACh; A0132; Sigma) were administered. Penicillin antibiotic (B151226; Shandong Lukang Pharmaceutical Co., Ltd., Jining, China) was administered after surgery. The concentration, doses, and administration methods were 1) urethane: 20%, 8 mL/kg for rats and 5 mL/kg for mice, I.P.; 2) clenbuterol: 0.2%, maintenance dose of 80 μL/kg/min, intravenous (I.V.); 3) propranolol: 0.4%, initial dose of 1.0 mL/kg and maintenance dose of 40 μL/kg/min, I.V.; 4) acetylcholine: 0.1%, maintenance dose of 20 μL/kg/min, I.V.; 5) atropine: 0.2%, initial dose of 0.8 mL/kg and maintenance dose of 40 μL/kg/min, I.V.; and 6) penicillin: 8 × 10⁵ IU dissolved in 2 mL saline, 0.4 mL/d per rat, intramuscular (I.M.).

This study was approved by the Wuhan University Animal Care and Use Committee and carried out complying with the Wuhan University Animal Use Guidelines. Adult (8–10 week old) male C57BL/6‐Il17a^tm1Bcgen mice, that is, IL‐17A‐EGFP transgenic mice were obtained from Beijing Biocytogen Inc. The model was established based on a published protocol with minor modifications.⁸ Briefly, each mice was intraperitoneally injected with 1 mg of urethane (Sigma‐Aldrich U2500, freshly prepared in 0.9% saline) per gram of body weight on Monday and Thursday every week for 10 weeks. Mice were then euthanized by CO₂ inhalation and subjected to intracardiac perfusion with 5 ml of ice‐cold phosphate‐buffered saline (PBS). After this, the lung was taken and placed in a 35‐mm petri dish containing 1 ml of ice‐cold (PBS) for further processing.

To detect activation of Glra3-expressing cells following sensory stimulation, adult C57BL/6J mice (10–14 weeks old, 3 mice/stimulus, 15 mice in total) were initially anesthetized with 2 g/kg urethane (Sigma-Aldrich, catalog #U2500, 125 mg/ml in sterile saline) through intraperitoneal injection to minimize neuronal activity caused by prurito- and nocifensive behavior. To prevent eye damage and dehydration, Oftagel (Santen Oy) was applied to eyes, and the mouse was injected subcutaneously with 0.5 ml saline. To maintain body temperature, a glove filled with body temperature water, which was continuously replaced to sustain temperature, was placed next to the mouse. When the mouse had been fully anesthetized for 10 min, the mouse was subjected to the stimulus. For pruritic stimulations, the mice were injected subcutaneously into the right dorsolateral calf either with 10 µl saline (1 female and 2 males) or a pruritic substance: 20 µg compound 48/80 (Sigma-Aldrich, catalog #c2313, dissolved in sterile saline, 1 female and 2 males) or 20 mM chloroquine (Sigma-Aldrich, catalog #PHR1258, dissolved in sterile saline, 1 female and 2 males).

Rats were anesthetized intraperitoneally with urethane (10%, 2 g/kg, U2500, Sigma-Aldrich, Zwijndrecht, The Netherlands). Anesthetic depth was assessed every 30 min by the absence of protective eye (corneal) reflexes and withdrawal reflexes after toe pinch. Rectal temperature was monitored throughout the experiment and was kept between 36.5 °C and 37.5 °C using a heating pad and blankets. During the surgical procedure, rats were tracheotomized and a tracheal cannula was placed and fixed with ligatures. Subsequently, rats were administered 100 μL—containing 50 μg/kg lipopolysaccharide (LPS) (Sigma-Aldrich, Zwijndrecht, The Netherlands)—intratracheally through the previously placed tracheal cannula, to induce acute lung injury. Three experimental procedures (Model A, B and C) were designed to investigate the role of the vagus nerve and high-fat nutrition on LPS-induced lung injury. A schematic overview of the experiments is shown in Figure 1A and applies to all models. Rats were euthanized 300 min after LPS administration by an overdose of intraperitoneally injected pentobarbital (150 mg/kg) (Euthesate™, Ceva Santé Animale, Naaldwijk, The Netherlands). Pulmonary tissue, bronchoalveolar fluid (BALF) and blood samples were isolated and examined in accordance with the American Thoracic Society guidelines [33 (link)].

Cystometry was performed as described previously15 31 (link)32 . The rats were anesthetized with urethane (1.2 g/kg, subcutaneously, sigma, U2500). A PE 50 catheter was inserted into the bladder for saline infusion at body temperature (37–38 °C) at a rate of 8 ml/h, and the intravesical pressure was continuously recorded. After stabilization for 30 minutes, the urodynamic parameters, including basal pressure (BP), maximum pressure (MP), micturition frequency (MF), intercontraction interval (ICI), and bladder capacity (BC), were recorded.