Bacteria in Atherosclerotic Plaques and Adverse Events

Research Objectives

1. Primary research objectives This project aims to establish a registry center for patients and specimen banks following carotid endarterectomy. Conduct plaque detection and analysis in patients undergoing carotid endarterectomy over a defined period, perform follow-up monitoring, track blood laboratory indicators, execute data cleansing, analyze bacterial species within plaques, identify bacteria associated with postoperative adverse events, and derive corresponding expected conclusions.
2. Secondary research objectives
3. Assess bacterial species within carotid plaques; identify bacterial differences and perform functional analysis in atherosclerotic plaques across distinct disease types.
4. Detect bacterial species within plaques associated with adverse postoperative events (cerebral infarction, carotid restenosis, death), identify risk bacteria linked to poor outcomes, and perform functional analysis.
5. Cleanse clinical data, analyze the correlation between postoperative adverse events and bacteria within high-risk plaques, and adjust for confounding factors including age, gender, body mass index, total cholesterol, high-density lipoprotein, low-density lipoprotein cholesterol, triglycerides, creatinine, diabetes, and hypertension to enhance the reliability of research findings.

Overall Design This study is a prospective, multicenter cohort study in western China. This registration study was primarily conducted in the Department of Neurosurgery at Tangdu Hospital, with participation from the Departments of Neurosurgery at the Second Affiliated Hospital of Xi'an Medical University, Hanzhong Central Hospital, Hanzhong 3201 Hospital, Baoji People's Hospital, and Pucheng County People's Hospital. The study population consisted of patients aged 18 to 90 years who underwent CEA surgery. Patients who meet all inclusion criteria and none of the exclusion criteria may be considered for enrollment after demonstrating informed consent to participate in this registry study and agreeing to sign the informed consent form.

All patients participating in this study underwent a comprehensive standardized evaluation conducted by a multidisciplinary team comprising neurologists, neurosurgeons, hematologists, endocrinologists, and other practitioners. The assessment included examinations of neurological, neuropsychological, and psychiatric conditions, endocrine disorders, and hematological parameters. Bacterial species identification was performed using 16SrDNA sequencing, with further validation through transmission electron microscopy.

Selected patients will undergo routine surgical treatment and attend follow-up appointments as recommended by their physicians, while accurately recording relevant data generated during clinical practice. Data information from patients' clinical visits will be documented, including preoperative assessment, preoperative care, operating room care, anesthesia management, intraoperative procedures, postoperative medical management, postoperative nursing care, and follow-up information.

Inclusion Criteria

1. Patients clinically diagnosed with moderate to severe carotid artery stenosis
2. Age 18 ≤ age ≤ 90
3. Patients undergoing carotid endarterectomy (CEA)
4. Preoperative mRS score for stroke patients: 0-2
5. Preoperative NIHSS score for stroke patients: 0-20
6. Patient's Glasgow Coma Scale (GCS) score upon admission: 8-15 points
7. No fever or evidence of infection at admission
8. Informed consent signed by the subject or their legal representative
9. Good compliance with follow-up visits

Exclusion Criteria

1. Brain tumor (presence of mass effect)
2. Patient's Glasgow Coma Scale (GCS) score < 8
3. Refractory hypertension unresponsive to medication (defined as sustained systolic blood pressure >185 mmHg or diastolic blood pressure >110 mmHg)
4. History of severe infection within the past three months
5. Severe or acute heart failure
6. Acute myocardial infarction or severe arrhythmia
7. Undergone major organ surgery or biopsy within the past month
8. Any active bleeding or recent bleeding (gastrointestinal, urinary tract, etc.) within the past month
9. Currently undergoing hemodialysis or peritoneal dialysis; known severe renal impairment (glomerular filtration rate < 220 mmol/L (2.5 mg/dL))
10. Concurrent malignant tumors, severe cardiopulmonary disease, or other conditions rendering the patient unable to tolerate surgery
11. Has participated in other interventional clinical studies that may influence the outcome assessment
12. Severe liver function abnormalities
13. Other circumstances deemed by the investigator to make participation in this study inappropriate or pose significant risk to the patient (e.g., inability to understand and/or comply with study procedures and/or follow-up due to psychiatric, cognitive, or emotional disorders)
14. Currently receiving immunosuppressive agents or undergoing immunotherapy
15. Severe chest or abdominal trauma requiring surgery、Severe Traumatic Brain Injury
16. Infectious Diseases such as Syphilis, AIDS, Hepatitis, and Tuberculosis
17. Patients with both hypertension and diabetes

Research Design Plan

1. Research Methodology: This study is a prospective, multicenter cohort study. This registry study is primarily conducted in the Department of Neurosurgery at Tangdu Hospital. The target population consists of adults aged 18 to 90 years who present to the neurosurgery department and require carotid endarterectomy. Patients meeting all inclusion criteria and none of the exclusion criteria may be considered for enrollment after demonstrating informed consent to participate in this registry study and agreeing to sign the informed consent form.
2. All patients participating in this study underwent a comprehensive standardized evaluation conducted by a multidisciplinary team comprising neurologists, neurosurgeons, hematologists, endocrinologists, and other practitioners. The evaluation included examinations of neurological status, neuropsychological and psychiatric conditions, endocrine disorders, and hematological parameters.
3. Patients enrolled in the study will undergo routine surgical treatment and attend follow-up visits as recommended by their physicians, while accurately recording all relevant data generated during clinical practice. Data information from patients' clinical visits will be documented, including preoperative assessment, preoperative care, operating room care, anesthesia management, intraoperative procedures, postoperative medical management, postoperative nursing care, and follow-up information.

Major Research Projects and Content

1. Assess the types of bacteria present in carotid plaques to preliminarily identify the relationship between bacteria and plaque associated with different disease types;
2. Detect bacteria within plaques associated with adverse postoperative outcomes (cerebral infarction, carotid restenosis, death), identify risk bacteria linked to poor outcomes, and conduct preliminary functional analysis of these bacteria.
3. Analyze clinical data to investigate the correlation between postoperative adverse events and bacteria within high-risk plaques, while adjusting for confounding factors such as age, gender, body mass index, total cholesterol, high-density lipoprotein, low-density lipoprotein, cholesterol, triglycerides, creatinine, diabetes, and hypertension to enhance the reliability of the study findings.

Key Statistical Indicators

This study will collect all data during the inpatient period (baseline phase) for patients requiring surgical intervention, as well as follow-up data after discharge. Data points to be collected for patients with carotid stenosis include:

1. Baseline Information Collection Admission Assessment and Enrollment: Demographic Data: Date of Birth, Gender, Educational Attainment, Ethnicity, Occupation Type, Health Insurance Type, Nature of Employment; Carotid Artery Stenosis Assessment: Document the degree of carotid artery stenosis in the patient; Preoperative Preparation: Past medical history and current medical history, including hypertension, diabetes, hyperlipidemia, cerebral infarction, renal disease, history of cardiovascular and cerebrovascular diseases, and other chronic conditions. Whether antibiotics were administered during surgery, types and reasons for previous surgeries, surgical sites, and use of analgesics postoperatively. Additionally, collect history of previous drug therapy, including medications used to treat and control related comorbidities. Physical Examination: Nutritional Assessment Status: Including height, weight, body fat percentage, muscle mass, grip strength; heart rate, respiratory rate, body temperature, systolic/diastolic blood pressure; Assessment of Positive Findings in Neurological Examination: Optic nerve damage, oculomotor nerve damage, limb paralysis, neck resistance; Comprehensive Patient Assessment and Optimization: Whether relevant assessments and optimization measures have been conducted, including water and electrolyte balance, complete blood count, biochemical tests, coagulation function tests, and status of vital organ function (lungs, heart, kidneys, liver). Scale Assessment: Evaluate patients' functional status preoperatively, intraoperatively, and postoperatively using the American Society of Anesthesiologists (ASA) risk classification, long-term quality of life assessment, and the General Hospital Anxiety/Depression Scale (GHAS), Depression Scale, NRS2002 score, VTE Caprini Risk Assessment Scale, Autar DVT Risk Scale, Operating Room Pressure Ulcer Risk Assessment (Braden), PONV Risk Score for preventing postoperative nausea and vomiting, and Visual Analogue Scale (VAS) for nausea and vomiting. Preoperative Communication: Patients receive counseling and guidance regarding the surgical and anesthetic procedures prior to surgery. Whether the patient received counseling and guidance from the surgeon, anesthesiologist, and nurse/head nurse regarding the surgical and anesthetic procedures. Record the duration of the communication visit (in minutes). Anti-hypertensive drug therapy: Initiate with low doses, prioritize long-acting formulations, employ combination therapy, and tailor treatment to individual needs. Common anti-hypertensive agents include five classes: beta-blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and diuretics, as well as fixed-dose combination formulations comprising these drugs. In the absence of concomitant vascular stenosis, post-CEA surgery blood pressure control below 140/90 mmHg is recommended. Diabetes Medication Management: Patients with carotid artery stenosis and concomitant diabetes must intensify dietary control. Target blood glucose levels: non-fasting glucose below 11.1 mmol/L, with HbA1c <7% during treatment. Lipid-lowering medication: Statin therapy is recommended for patients with carotid stenosis. For those at high risk of stroke, low-density lipoprotein (LDL) levels should be controlled below 100 mg/dL. Niacin or fibrate-based lipid-lowering agents may be considered for patients with hypertriglyceridemia. Smoking Cessation: Quitting smoking is a critical preventive and therapeutic measure for carotid stenosis. Smokers should be strictly required and encouraged to quit, and exposure to secondhand smoke should be avoided. Anti-platelet and Anticoagulant Therapy: Recommended anti-platelet agents include aspirin and clopidogrel. Low-dose aspirin (75-150 mg/day) achieves equivalent efficacy to high-dose regimens. Heparin therapy should be administered prior to carotid artery occlusion during surgery. Hyperhomocysteinemia management: Elevated homocysteine levels increase stroke risk. Patients with carotid stenosis and concomitant hyperhomocysteinemia should receive B-complex vitamin therapy preoperatively.
2. Preoperative Evaluation Indications for Surgery: Absolute Indications: Symptomatic carotid stenosis with noninvasive testing showing ≥70% stenosis or angiography revealing ≥50% stenosis. Relative Indications: (1) Asymptomatic carotid stenosis with noninvasive testing showing ≥70% stenosis or angiography revealing ≥60% stenosis; (2) Asymptomatic carotid stenosis with noninvasive testing showing <70% stenosis, but angiography or other studies indicating unstable stenotic lesions; (3) Symptomatic carotid stenosis with noninvasive testing showing 50%-69% stenosis.

Perioperative Pharmacotherapy: All enrolled patients are recommended to receive single antiplatelet therapy with aspirin (100 mg/day) or clopidogrel (75 mg/day) preoperatively to reduce thrombosis risk. High-dose antiplatelet agents are not recommended. Intraoperatively, administer heparin anticoagulation 5 minutes prior to arterial occlusion to prolong activated clotting time (ACT) or activated partial thromboplastin time (aPTT) by at least 1.5-fold. Postoperatively, continue single antiplatelet therapy for at least 4 weeks.

Timing of Surgery: (1) For acute ischemic stroke, surgery is relatively safe after 6 weeks post-onset. However, for patients with recent symptom onset and imaging evidence of unstable plaques, early intervention should be pursued whenever possible, ideally within 2 weeks. (2) For patients with TIA or minor stroke, intervention may be performed within 2 weeks of the event if there are no contraindications to early revascularization. (3) In cases of bilateral lesions, the interval between bilateral surgeries may range from 2 to 4 weeks based on clinical circumstances, with priority given to the symptomatic side and/or the side with more severe stenosis.

Choice of surgical approach: Includes two types: the eversion endometrial resection and the traditional longitudinal incision endometrial resection.

Complications and Prevention: (1) Stroke and Mortality. Stroke is associated with ischemia caused by plaque detachment and occlusion. Strict individualized perioperative blood pressure management is generally required. Intraoperative close monitoring is essential to reduce strokes due to hemodynamic disturbances. Intraoperative cerebral oxygen saturation monitoring, TCD monitoring, and intraoperative electrophysiological monitoring should be performed. Intraoperative stump pressure should be monitored. Perform gentle intraoperative maneuvers and selectively use bypass tubes. Anticoagulant therapy may be administered based on specific circumstances. Perioperative measures such as antiplatelet agents should be employed to reduce embolic risk. (2) Cranial Nerve Injury. The most commonly affected nerves include the hypoglossal, vagus, and accessory nerves, with injuries typically being transient. These may be related to surgical traction and edema. Intraoperative maneuvers should be gentle, with precise identification of delicate cervical anatomical structures to avoid nerve injury from surgical traction. Cutaneous nerve injury is generally difficult to prevent. Postoperative numbness around the jaw or behind the ear may occur. For all enrolled surgical patients, the length of the cervical surgical incision should be controlled at approximately 3-5 cm to minimize cutaneous nerve injury caused by excessive incision length. (3) Hyperperfusion Syndrome. Primary clinical manifestations include severe localized headache, localized and/or generalized convulsions, and cerebral hemorrhage in the ipsilateral hemisphere. Prophylactic administration of antihypertensive and dehydrating agents (e.g., mannitol) during carotid artery reperfusion and postoperatively may mitigate cerebral edema. (4) Neck hematoma and laryngeal edema. The former is often associated with incomplete local hemostasis or inadequate arterial suturing, while the latter may relate to anesthesia intubation. Closely monitor oxygen saturation, enhance suturing techniques, and meticulously control bleeding-particularly preventing damage to extensive veins and lymph nodes during dissection. Once hematoma or laryngeal edema occurs, prevent asphyxia. (5) Thrombosis and restenosis. Contributing factors include intraoperative mishandling, inadequate postoperative medication, and excessive smooth muscle or intimal hyperplasia. Monitor for heparin resistance. Administer perioperative oral antiplatelet agents and intimal hyperplasia inhibitors; consider traditional Chinese medicine when indicated.

Data Collection and Definition: Patient demographic and clinical data, including age, gender, smoking and drinking history, past medical history, echocardiography results, angiography assessment findings, and medications used at discharge, were collected from electronic medical record systems by physicians unaware of the study's purpose. All participants provided fasting (>8 h) peripheral venous blood for measurement of white blood cell count (WCC), platelet count (PLT), hemoglobin (HGB), fasting blood glucose (FBG), total bilirubin (TB), direct bilirubin (DBIL), indirect bilirubin (IBIL), creatine kinase (CK), creatine kinase isoenzyme (CK-MB), creatinine, uric acid (UA), high-sensitivity C-reactive protein (Hs-CRP), and lipid profile (total cholesterol (CHO), TG, LDL, high-density lipoprotein (HDL), lipoprotein a (Lpa)).

Intraoperative Management Operating Room Nursing: Operating room parameters (temperature, humidity), entry/exit times, duration of surgery, level of consciousness upon entry, psychological status, catheterization method, antibiotic prophylaxis and allergic reactions, infusion reactions, hypothermia prevention status confirmation (including body temperature, use of warming blankets, infusion of fluids, use of irrigation solutions), VTE (Venous Thromboembolism) prevention and pressure ulcer prevention (including intraoperative positioning, care of pressure points, and care frequency); Pre-anesthesia management: Including anesthesia time, anesthesia method, monitoring parameters (anesthesia depth, cardiac output, neuromuscular blockade, temperature, arterial blood gas, etc.), presence of preoperative local anesthesia at incision site, presence of postoperative local anesthesia at incision site, intraoperative circulatory status, fluid output, fluid replacement volume, blood transfusion volume, transfusion reactions, level of consciousness grading, Steward recovery score, and extubation status prior to leaving the operating room. Also includes pre-anesthesia medication administration, recording brand name, generic name, dosage, frequency, and duration; Anesthetic induction: Record patient cardiopulmonary and renal function data. Routinely monitor electrocardiogram (ECG), invasive mean arterial pressure (IMAP), oxyhemoglobin saturation (SpO₂), partial pressure of carbon dioxide in end-tidal gas (PetCO₂), Bispectral Index (BIS). Document anesthetic induction medications, including brand name, generic name, dosage, frequency, and duration.

Anesthesia Maintenance Protocol: Record anesthesia maintenance protocol data, including drug brand names, generic names, dosages, frequency, and duration; Blood Pressure Management: Document whether the patient experienced blood pressure fluctuations during surgery, including deviations exceeding ±20% from baseline levels and SBP ≥ 180 mmHg. Record corresponding blood pressure management medications, including brand names, generic names, dosages, frequency, and duration; Fluid Therapy: Document fluid administration methods during surgery, including vasoactive agents, normal saline, sodium acetate Ringer's solution, and Ringer's lactate solution. Record events where plasma osmolarity or stroke volume variation (SVV) monitoring exceeded ≥13%.

Ventilation Strategy: Document data points related to the ventilation strategy employed, including associated events.

Temperature Monitoring and Warming: Prevent hypothermia during surgery. Document episodes of core temperature ≤36°C.

Thromboprophylaxis: Include thromboprophylactic measures, medications, or adjunctive products. Document occurrence of postoperative venous thromboembolic events.

CEA Surgical Procedure Flowchart: All surgeries in this study were performed by Associate Professor Liu Bei as the primary surgeon. Intraoperative cerebral oxygenation monitoring, electrophysiological monitoring, and transcranial Doppler ultrasound monitoring were performed. The patient was positioned supine with the head hyperextended, tilted backward, and deviated toward the contralateral side. Marked an anterior sternocleidomastoid muscle incision approximately 3-5 cm in length. Following routine disinfection and draping, sequentially incised the skin, subcutaneous tissue, and platysma muscle. Make a sharp dissection along the anterior border of the sternocleidomastoid muscle to deeper structures, dissecting and exposing the carotid sheath. Following exposure of the carotid sheath, administer heparin at a dose of 83 IU/kg. Utilized specialized CEA instruments to dissect the carotid sheath, progressively exposing the common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA). Administer lidocaine block at the carotid sinus location at the bifurcation site. Sequentially occlude the superior thyroid artery, internal carotid artery, external carotid artery, and common carotid artery, controlling arterial occlusion time between 10-20 minutes. Under microscopic visualization, make a longitudinal incision in the artery. Carefully dissect and remove the plaque. Irrigate repeatedly with heparinized saline. Microscopically remove any residual plaque. After confirming a smooth intima, perform continuous suturing from the distal ICA toward the CCA at approximately 1 mm intervals. Following the procedure, perform intraoperative fluorescein angiography to assess vascular patency.

Postoperative Management Secondary prevention strategies for postoperative patients should be implemented according to current guidelines. Maintain activated clotting time (>24 hours) with unfractionated heparin (83 IU/kg, titrated as needed) postoperatively.

Postoperative Diet: Document time to clear liquid intake, time to clear liquid diet, and time to regular diet following surgery.

Pain Management: Record location, nature, intensity score, duration, analgesic usage, and use of analgesic pumps. Conduct daily pain assessments throughout hospitalization until discharge, documenting the highest pain score recorded that day.

Respiratory Management: Document tracheal intubation status and extubation time; record postoperative use of intravenous (pre-ambulation) and nebulized medications; Gastrointestinal Management: Includes mucosal protection measures and mucosal lesions; Imaging Follow-up: Document results of follow-up imaging studies, including head/chest CT, head MRI, head and neck CTA; Other Postoperative Management Principles: Including parenteral nutrition, time of postoperative fluid cessation, fluid reactions, nutritional assessment at discharge, mental status at discharge, KPS score at discharge, and other scale assessments; inpatient parameters (including hospitalization costs, total hospitalization days, postoperative hospitalization days). If secondary surgery occurs during hospitalization, corresponding records must be made; Satisfaction: Assess patient satisfaction with clinical services at discharge; Patch collection and preservation: Atherosclerotic plaque samples excised from the carotid bifurcation during endarterectomy are collected in Eppendorf tubes, rapidly frozen in liquid nitrogen, stored at -80°C, or fixed in 2.5% glutaraldehyde electron microscopy fixative for subsequent analysis.

Pharmacotherapy Patients undergoing perioperative medication therapy, including traditional Chinese medicine or adjunctive devices, require documentation of the following: generic name, brand name, dosage and administration, frequency of administration, duration of use, purpose of medication, drug cost, and reason for medication change. As surgical management is process-oriented, medication therapy should be categorized according to clinical practice (admission, preoperative, intraoperative, postoperative, discharge) and summarized in a master table. Reasons for medication adjustments must be recorded. Poor efficacy: Determined based on surgical outcome, e.g., non-healing incision. Significant medical events/adverse events/adverse reactions: Gastrointestinal reactions; edema; weight gain; changes in liver/kidney function; allergies; other discomforts. Researcher-initiated adjustments, patient-initiated adjustments (specific reasons).

Adverse Event (AE)/Serious Adverse Event (SAE):

This project reports adverse events/serious adverse events as specified in the protocol. All adverse reaction events must be collected and reported, regardless of whether they are related to these products. Since surgical management is process-based, AEs/SAEs are categorized and summarized by clinical pathway in the master table.

Follow-up Period Data Collection Follow-up: Post-discharge follow-up schedule, conducted as outpatient visits. Follow-up content includes: General patient status, including physical examination and necessary laboratory tests, required imaging studies, and relevant scale assessments, including neuropsychiatric scoring. If the patient undergoes secondary surgery or is readmitted during follow-up, corresponding records must be documented. If additional laboratory tests are performed during follow-up, copies of the original reports must be filed in the medical record. The patient's current medical history, hospitalization details, medication status-particularly reasons for medication changes, dosages, and efficacy-must be documented. Investigators must enter laboratory test data into the eCRF (electronic Case Report Form) and retain the original test reports. Investigators must also provide assessments of laboratory test results as normal/abnormal and reference ranges.

Research time-frame

This study will be conducted in the following two phases:

1. Study Preparation: This phase includes developing materials (CRF, EDC), Informed Consent Forms (ICF), selecting and evaluating study sites, obtaining ethics committee approval, developing the EDC system, and initiating the protocol and study sites. This phase is expected to take 2 months.
2. Patient enrollment, testing, follow-up, and data analysis completion: Estimated at 16 months.

The total duration of this study, from initiation to completion of the final study report, is projected to be 18 months.