This study employed a prospective, controlled, parallel-group clinical trial design with equal randomization, conducted at Ningxia Medical University General Hospital affiliated Cancer Hospital. The study received ethical approval from the Ethic Committee of Ningxia Medical University General Hospital (approval number: KYLL-2023-0045) and was registered a priori with the Chinese Clinical Trial Registry (www.chictr.org.cn, ChiCTR2300068563; 23/02/2023). The enrollment period spanned from February 2023 to October 2023. We used the CONSORT checklist when writing our report [16].

Adhering to Good Clinical Practice guidelines and the Declaration of Helsinki, written informed consent was obtained from participating patients or authorized surrogates. The trial considered ethical principles to protect patient rights and well-being.

Inclusion criteria encompassed patients aged 18–70 years, ASA physical status 1–3, with primary or invasive breast cancer without distant metastasis (stage 0 to III), and with or without axillary lymph node dissection. Exclusion criteria included allergies to study substances, diabetes, coronary heart disease, chronic inflammatory diseases, previous surgical history of breast cancer (except diagnostic biopsy), neuropsychiatric diseases hindering informed consent, incapacity to understand the study protocol or refusal to participate, and regular usage of corticosteroids or anti-inflammatory drugs.

Patients in this study were allocated to one of four groups using a computer-generated randomization process with a ratio of 1:1:1:1. To ensure blinding, group assignments and patient study numbers were concealed within opaque, sealed envelopes. These envelopes were opened only after patients signed a written informed consent form preoperatively.

The experimental groups included sevoflurane anesthesia (S), sevoflurane anesthesia plus intravenous lidocaine (SL), propofol total intravenous anesthesia (P), and propofol total intravenous anesthesia plus intravenous lidocaine (PL). The selection of these anesthesia methods was based on their relevance to our research question and their common use in breast cancer surgery.

For safety reasons, the attending anesthesiologist used non-blind criteria throughout the lidocaine infusion. To maintain the integrity of the blinding process, researchers involved in postoperative follow-up, blood collection, laboratory testing, data analysis, and interpretation remained unaware of the grouping.

Non-lidocaine groups received a placebo infusion of saline.

Patients in this study underwent routine fasting for 8 h and were restricted from drinking 4 h prior to surgery. Additionally, 30 min before anesthesia, patients received an intramuscular injection of pentylenetetrazol hydrochloride (1 mg). This pre-anesthetic medication aims to inhibit salivary gland and airway gland secretion.

During anesthesia induction, all patients across the four groups (S, SL, P, PL) received a standardized regimen consisting of intravenous midazolam (0.05 mg kg−1), sufentanil (0.3 µg kg−1), etomidate (0.3 mg kg−1), and rocuronium (0.6 mg kg−1). Following laryngeal mask placement, mechanical ventilation was employed to maintain the end-expiratory carbon dioxide concentration at 35–45 mmHg with a 50/50 mixture of O2/air at a flow rate of 2 L/min.

For the propofol total intravenous anesthesia (TIVA) groups (P and PL), anesthesia maintenance involved a constant infusion of propofol (3–5 mg kg−1 h−1) and remifentanil (0.3 µg kg−1 min−1) to sustain a bispectral index (BIS) within the range of 40–60, ensuring optimal analgesia.

In the sevoflurane groups (S and SL), anesthesia maintenance included continuous inhalation of 1-3% sevoflurane (1-1.5 MAC) to maintain BIS values between 40 and 60. Simultaneously, remifentanil was continuously administered at 0.3 µg kg-1 min-1 for intraoperative analgesia. Intraoperatively, mean arterial pressure was maintained within 20% of the basal value, and the use of vasoactive drugs (e.g., ephedrine) was determined by the attending anesthesiologist.

For the lidocaine groups (PL and SL), a loading dose of 1% lidocaine (1.5 mg kg−1) was administered during induction, followed by a continuous infusion of lidocaine at 2 mg kg−1 h−1 throughout the procedure. Groups P and S received an equal volume of saline instead of lidocaine.

Although the dosing protocols maintained plasma concentrations below toxic levels, a 20% lipid emulsion was prepared in the operating room as a safety precaution. Anesthesiologists were informed of its location. Midazolam (0.05 mg kg−1) was administered for local anesthetic poisoning seizures. If symptoms persisted or the patient’s condition was unstable, a rapid intravenous loading dose of 20% lipid emulsion (1.5 ml kg−1) was given, followed by continuous micropump infusion at a rate of 0.25 ml kg−1 min−1. The loading dose could be repeated (up to three times), and the infusion rate could be increased, not exceeding 0.5 ml kg−1 min−1.

At the end of surgery, neuromuscular antagonism was achieved through the administration of neostigmine (1 mg) and atropine (0.5 mg). Postoperative analgesia included the initial choice of acetaminophen (1 g), administered when patients reported pain in the post-anesthesia care unit and ward.

The primary outcomes encompassed serum concentrations of H3Cit, MPO, NE, MMP-9, and VEGF-A. Venous blood samples (5 ml) were collected from each patient before anesthesia induction and 3 h post-operation [17], using serum separator tubes (KWS, Hebei, China). This collection, performed from a different venous access site and then used for drug administration, aimed to collect blood samples during the peak release of neutrophil extracellular trapping markers and to reduce instances of patient culling resulting from self-administered oral medication. Post-collection, blood was centrifuged at 3000 times per minute for 20 min at 4–6 °C within 3 h. The resulting supernatants were transferred into 2 ml aliquots at -80 °C for subsequent enzyme-linked immunosorbent assay (ELISA) analysis.

ELISA measurements were conducted using commercially available kits for H3Cit, MPO, NE, MMP-9, and VEGF-A (Jianglai Bio and Boster, China) following the manufacturer’s instructions.

The sandwich ELISA technique determined MPO levels, utilizing 96-well plates pre-coated with anti-MPO antibodies. A biotin-conjugated anti-MPO antibody and horseradish peroxidase (HRP)-streptavidin conjugate (SABC solution) facilitated the detection. The HRP substrate, 3,3’,5,5’-Tetramethylbenzidine (TMB), initiated the color change. In brief, serum samples were thawed and diluted (1:10) with sample dilution buffer. Test sample dilutions (0.1 ml aliquots) were added to wells, sealed, and incubated at 37 °C. Subsequent steps included the addition of a biotin-conjugated detection antibody, SABC working solution, and TMB substrate. Absorbance at 450 nm was measured using the ELISA Thermo Scientific™ Multiskan™ FC Enzyme Labeler.

The serum concentration of each factor was determined from standard and control samples, with curves drawn on coordinate paper. For accuracy, each factor underwent a repeat measurement. Inter-batch and intra-assay coefficients of variation were assessed, with values meeting the manufacturer’s specifications.

Perioperative data, including patient and breast tumor characteristics, anesthetic and surgical factors, and intraoperative details, were also collected. This additional information provides a comprehensive understanding of the study context. These meticulous procedures ensure the robustness of our data collection and analysis, contributing to the reliability of our study outcomes.

In anticipation of a scientifically significant reduction of 2.0 ng ml-1, approximately 20% lower than the typical serum estimations of NETosis MPO values (10–15 ng ml-1 with a standard deviation of 3 ng ml-1), we conducted a power analysis. Assuming a type I error of 0.05 and a type II error of 0.1, a sample size of n = 25 patients per group would yield 90% power to detect this anticipated difference. To account for potential missing data, we enrolled n = 30 patients in each group.

For statistical analysis, GraphPad Prism TM v9 was employed. Data normality and homogeneity were assessed. Normally distributed and homogenous data were subjected to analysis of variance (ANOVA) with post hoc Bonferroni correction for intergroup comparisons. The choice of ANOVA aimed to capture differences between independent groups effectively.

Within-group differences in serum marker values before and after anesthesia and surgery for normally distributed data were assessed using paired Student’s t-tests. Skewed or uneven variances data were analyzed using the Kruskal-Wallis H test with post hoc Bonferroni correction for group comparisons, while within-group differences were assessed using the Wilcoxon test.

Categorical variables underwent analysis using chi-square tests, continuity correction chi-square tests, or Fisher exact tests, depending on the nature of the data. Data presentation followed standard conventions: mean (standard deviation), median (25–75% interquartile range), or n (%).

GraphPad Prism TM v9 was chosen for its suitability for biomedical research and its user-friendly interface. A threshold of P < 0.05 was considered statistically significant. This robust statistical approach ensures a comprehensive analysis of our data, aligning with the scientific rigor required for meaningful interpretation and drawing valid conclusions from our study.