Cerebrospinal Fluid Mitochondrial Biomarkers in Ischemic Stroke and Alzheimer Disease

Capital Medical University40 enrolled

Overview

This prospective observational study aims to investigate cerebrospinal fluid mitochondrial biomarkers in patients with ischemic stroke and Alzheimer disease controls who undergo diagnostic lumbar puncture for clinical indications at Xuanwu Hospital, Capital Medical University. Residual cerebrospinal fluid samples will be analyzed by flow cytometry to quantify mitochondrial content, mitochondrial membrane potential, and cellular or vesicular source-related markers. The flow cytometry panel will include MitoTracker, JC-1, and membrane-associated markers including CD45, CD41, CD24, vWF, and EAAT1. In patients with ischemic stroke, the study will further examine whether cerebrospinal fluid mitochondrial measurements are associated with neurological severity and functional outcomes, including admission and discharge NIHSS scores and the 90-day modified Rankin Scale score. Alzheimer disease patients undergoing diagnostic lumbar puncture will serve as disease controls for biomarker comparison.

Mitochondrial dysfunction is closely associated with ischemic brain injury, but the clinical relevance of extracellular or cell-associated mitochondrial signals in the cerebrospinal fluid of patients with ischemic stroke remains insufficiently characterized. Cerebrospinal fluid may provide a biologically informative compartment for assessing mitochondrial injury, mitochondrial membrane potential, and potential cellular sources of mitochondrial signals in neurological diseases. This is a single-center, prospective, observational study conducted at Xuanwu Hospital, Capital Medical University. The study will enroll patients with ischemic stroke or Alzheimer disease who require diagnostic lumbar puncture as part of routine clinical evaluation between March 2026 and May 2026. No lumbar puncture will be performed solely for research purposes. After completion of clinically required testing, available residual cerebrospinal fluid will be collected for research flow cytometry analysis. Flow cytometry will be used to evaluate cerebrospinal fluid mitochondrial content and related phenotypic features. MitoTracker will be used to detect mitochondrial signal, JC-1 will be used to assess mitochondrial membrane potential, and membrane-associated markers including CD45, CD41, CD24, vWF, and EAAT1 will be used to characterize potential leukocyte-associated, platelet-associated, neuronal/extracellular vesicle-associated, endothelial-associated, and astrocytic/glial-associated components. The primary analysis will focus on patients with ischemic stroke and will evaluate the association between cerebrospinal fluid mitochondrial content and 90-day functional outcome measured by the modified Rankin Scale. Secondary analyses will examine associations between cerebrospinal fluid mitochondrial measurements and admission NIHSS, discharge NIHSS, and changes in NIHSS during hospitalization. Exploratory analyses will compare cerebrospinal fluid mitochondrial and membrane-marker profiles between ischemic stroke patients and Alzheimer disease controls.

Eligibility

Sex: ALLMin age: 18 Years

Medical Language ↔ Plain English

Inclusion Criteria: * Age 18 years or older. Patients treated at Xuanwu Hospital, Capital Medical University between March 2026 and May 2026. Diagnosis of ischemic stroke or Alzheimer disease according to standard clinical diagnostic criteria. Diagnostic lumbar puncture performed for clinical indications as part of routine medical care. Availability of residual cerebrospinal fluid after completion of clinically required testing. Ability to provide written informed consent, or availability of a legally authorized representative to provide consent when appropriate. For ischemic stroke patients, availability of baseline neurological assessment and planned follow-up for 90-day modified Rankin Scale assessment. Exclusion Criteria: * Lumbar puncture performed solely for research purposes rather than clinical indication. Insufficient residual cerebrospinal fluid volume for research flow cytometry analysis. Grossly bloody or severely contaminated cerebrospinal fluid sample that precludes reliable flow cytometry analysis. Known central nervous system infection, malignant meningitis, or other inflammatory or neoplastic condition that, in the investigator's judgment, may substantially confound cerebrospinal fluid mitochondrial measurements. Inability to obtain informed consent from the participant or legally authorized representative. Missing key clinical outcome data, including admission NIHSS, discharge NIHSS, or planned 90-day mRS follow-up for ischemic stroke participants. Any condition judged by the investigator to make the participant unsuitable for inclusion in the study.

Outcomes

Primary Outcomes

Association Between Cerebrospinal Fluid MitoTracker-Positive Event Count and 90-Day Modified Rankin Scale Score in Patients With Ischemic Stroke

The primary outcome is the association between cerebrospinal fluid (CSF) MitoTracker-positive event count measured at baseline and functional outcome assessed by the modified Rankin Scale (mRS) on Day 90 after stroke onset. The mRS ranges from 0 to 6, with higher scores indicating greater disability or death.

Time frame: Day 90 after stroke onset

Secondary Outcomes

Association Between Cerebrospinal Fluid MitoTracker Median Fluorescence Intensity and 90-Day Modified Rankin Scale Score in Patients With Ischemic Stroke

The association between cerebrospinal fluid (CSF) MitoTracker median fluorescence intensity measured at baseline and 90-day modified Rankin Scale (mRS) score will be assessed in patients with ischemic stroke.

Time frame: Day 90 after stroke onset

Association Between Cerebrospinal Fluid MitoTracker-Positive Event Count and Baseline National Institutes of Health Stroke Scale Score

The association between cerebrospinal fluid (CSF) MitoTracker-positive event count measured at baseline and neurological deficit severity will be assessed using the National Institutes of Health Stroke Scale (NIHSS) score obtained within 24 hours after hospital admission. Higher NIHSS scores indicate more severe neurological impairment.

Time frame: Baseline, within 24 hours after hospital admission

Association Between Cerebrospinal Fluid MitoTracker-Positive Event Count and Discharge National Institutes of Health Stroke Scale Score

The association between cerebrospinal fluid (CSF) MitoTracker-positive event count measured at baseline and neurological deficit severity at discharge will be assessed using the National Institutes of Health Stroke Scale (NIHSS) score recorded at hospital discharge.

Time frame: At hospital discharge, up to 30 days after hospital admission

Association Between Cerebrospinal Fluid MitoTracker-Positive Event Count and Change in National Institutes of Health Stroke Scale Score During Hospitalization

Change in National Institutes of Health Stroke Scale (NIHSS) score will be calculated as the discharge NIHSS score minus the baseline NIHSS score obtained within 24 hours after hospital admission. The association between cerebrospinal fluid (CSF) MitoTracker-positive event count measured at baseline and change in NIHSS score during hospitalization will be evaluated.

Time frame: From baseline within 24 hours after hospital admission to hospital discharge, up to 30 days after hospital admission

Cerebrospinal Fluid JC-1 Red-to-Green Fluorescence Ratio

Mitochondrial membrane potential in cerebrospinal fluid (CSF)-associated mitochondrial events will be assessed using JC-1 dye by flow cytometry. The JC-1 red-to-green fluorescence ratio will be used as a measure of mitochondrial polarization status.

Time frame: Baseline, at the time of diagnostic lumbar puncture

Difference in Cerebrospinal Fluid MitoTracker-Positive Event Count Between Patients With Ischemic Stroke and Alzheimer Disease Controls

Cerebrospinal fluid (CSF) MitoTracker-positive event count will be compared between patients with ischemic stroke and Alzheimer disease controls at baseline.