The study included patients who were candidates for atrial fibrillation catheter ablation and those scheduled for cardiac surgery with cardiopulmonary bypass, encompassing individuals with and without a history of stroke within the past year among AF patients. All patients took oral rivaroxaban anticoagulation (dosage determined by creatinine clearance rate, 20 mg qd or 15 mg qd).They were categorized into two groups based on stroke occurrence: the stroke-AF group and the non-stroke AF group. Blood samples were collected from patients undergoing AF catheter ablation, including samples from within the left atrial appendage(LAA) and peripheral veins(PV). LAA tissues were collected from AF patients scheduled for cardiac surgery with cardiopulmonary bypass. Relevant clinical data of the study participants were obtained, including age, height, gender, BMI, NYHA classification, complete blood count, liver and kidney function tests, D-Dimer, fibrinogen levels, echocardiography, cardiac CT, 12-lead ECG, as well as their medical history of prior cardiac surgeries, concurrent medical conditions, and medications. The research protocol was reviewed and approved by the ethics committee, and all participants with concurrent malignancies or autoimmune diseases, and patients with hyperthyroidism. The definition of ischemic atrial fibrillation-related provided written informed consent before entering the study.

The diagnosis of AF is established through a 12-lead electrocardiogram (ECG) and 24-h ambulatory ECG monitoring, encompassing both valvular and non-valvular atrial fibrillation. Exclusion criteria comprise patients with the following conditions: those requiring pacemaker therapy due to concomitant bradyarrhythmias, patients stroke is as follows: 1) a confirmed diagnosis of atrial fibrillation, 2) the occurrence of a stroke or transient ischemic attack (TIA) within one year (the diagnoses of stroke and TIA were established through outpatient interviews and CT/MRI imaging), 3) the absence of vascular-related stroke etiologies, such as intracranial or extracranial arterial stenosis (determined by cerebral vascular CTA).

For patients scheduled for AF catheter ablation, with blood samples collected from within the LAA and PV: 2% lidocaine local anesthetic was administered for right femoral vein puncture in the groin area. Subsequently, a Super Stiff Guidewire (Abbott Medical, 180 cm) and a Transseptal Guiding Introducer (Fast-Cath, Abbott Medical, 8.5F, 63 cm) were advanced through the femoral vein into the right atrium. Using the Fast-Cath, a Transseptal Needle (St. Jude Medical, 71 cm) was employed to puncture the interatrial septum. A PIG (Cordis, 6F, 110 cm) was then positioned deep within the LAA to collect 5 ml of blood from within the LAA, simultaneously with the collection of 5 ml of blood through the femoral vein. Subsequently, 10 ml of Iodixanol (32 mg/100 ml) was injected through the PIG, and fluoroscopy was employed to confirm the placement of the catheter tip deep within the LAA (Fig. 1).

Left atrial appendage angiography confirms PIG in LAA

For patients scheduled for surgical atrial fibrillation ablation under cardiopulmonary bypass,with left atrial appendage tissue collected: General anesthesia was administered during surgery, and cardiopulmonary bypass was established. The pericardium was opened using thoracoscopy, and a portion of the LAA tissue was isolated and excised after ligation. The harvested left atrial appendage tissue was rinsed with sterile injection water and fixed in 10% formaldehyde for subsequent analysis (Fig. 2).

Left atrial appendage tissue

The serum VCAM-1 concentration (pg/ml) was quantitatively analyzed using ELISA. Following blood sample collection, centrifugation was performed at 3000 rpm for 20 min, and the upper serum layer was collected and stored at -80 degrees Celsius. Serum VCAM-1 concentrations were determined for all samples using an enzyme-linked immunosorbent assay (ELISA) kit from Animal union Biotechnology, with intra-assay and inter-assay coefficients of variation of 4.5% and 8.9%, respectively.

Immunohistochemical assessment of VCAM-1 average optical density values (AOD) in LAA tissue was conducted. Left atrial appendage tissues fixed in 10% formaldehyde were prepared as paraffin sections. All tissue sections were stained with hematoxylin and eosin after being labeled with recombinant antibodies, where positive expression was indicated by brownish-yellow coloration. Images were acquired by scanning the slides using Omnipath Slide Center software, and subsequent analysis of AOD was performed using Image J software. (AOD was calculated as the optical density value (OD) divided by the area (Area)).

Statistical analysis was performed using SPSS 23.0 software (SPSS, Chicago, Illinois, USA). The normality of continuous variables was assessed using the Kolmogorov–Smirnov test, and these variables were presented as mean ± standard deviation (Mean ± SD). The analysis of continuous variables was conducted using the t-test. Risk factors for atrial fibrillation-related strokes were assessed through univariate analysis and multivariate logistic regression analysis. Receiver Operating Characteristic (ROC) curves were constructed, and the optimal cutoff values were selected using the Youden Index.