Deputy Director of Department of Neurosurgery, the Second Affiliated Hospital of Zhejiang University School of Medicine；Professor；
Conditions: Aging
Sponsor: Second Affiliated Hospital, School of Medicine, Zhejiang University

 
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Study Protocol 
1. Background 
Research on the relationship between biomarkers in human cerebrospinal fluid (CSF) 
and aging is a current hotspot in the field of neuroscience. Analyzing specific proteins and 
other molecules in CSF can provide important information for the early diagnosis and 
pathological progression of neurodegenerative diseases. Common biomarkers in CSF 
include amyloid-beta (Aβ42) and phosphorylated tau protein (p-tau181)1. The 
concentration changes of these markers have significant diagnostic value in Alzheimer's 
disease (AD) and other neurodegenerative diseases. A decrease in Aβ42 levels and an 
increase in p-tau181 levels are closely associated with the pathological features of AD2. 
Aging is a major risk factor for neurodegenerative diseases, and studies have found that the 
levels of Aβ and tau proteins in CSF undergo significant changes with increasing age. These 
changes are particularly evident in neurodegenerative diseases such as Alzheimer's disease 
and Lewy body dementia3. Research shows that monitoring biomarkers in CSF can aid in 
the early identification of these diseases, thereby facilitating early intervention and 
treatment4. Modern studies utilizing high-throughput proteomics and other advanced 
biotechnologies have revealed many new potential biomarkers in CSF. For example, mass 
spectrometry can detect protein changes related to neuroinflammation and blood-brain 
barrier function, which are crucial for understanding the neurodegenerative pathological 
mechanisms during aging5. 
In-depth research into CSF biomarkers not only aids in understanding the 
neurodegenerative changes during aging but also promotes the development of early 
diagnostic techniques, improving the management and treatment of neurological diseases 
in the elderly population. Future research will further explore the mechanisms and 
applications of these biomarkers, aiming to achieve broader and more precise diagnosis in 
clinical practice.   
References: 
1. 
Kurihara M, Matsubara T, Morimoto S, et al. Neuropathological changes 

 
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associated with aberrant cerebrospinal fluid p-tau181 and Aβ42 in Alzheimer's disease and 
other neurodegenerative diseases. Acta Neuropathol Commun. 2024;12(1):48. Published 
2024 Mar 27. doi:10.1186/s40478-024-01758-3 
2. 
Mattsson-Carlgren N, Grinberg LT, Boxer A, et al. Cerebrospinal Fluid 
Biomarkers in Autopsy-Confirmed Alzheimer Disease and Frontotemporal Lobar 
Degeneration. 
Neurology. 
2022;98(11):e1137-e1150. 
doi:10.1212/WNL.0000000000200040 
3. 
O'Leary K. A timeline of biomarker changes before Alzheimer's disease. Nat 
Med. Published online March 15, 2024. doi:10.1038/d41591-024-00018-0 
4. 
Jia J, Ning Y, Chen M, et al. Biomarker Changes during 20 Years Preceding 
Alzheimer's Disease. N Engl J Med. 2024;390(8):712-722. doi:10.1056/NEJMoa2310168 
5. 
Krance SH, Cogo-Moreira H, Rabin JS, Black SE, Swardfager W; Alzheimer's 
Disease Neuroimaging Initiative. Reciprocal Predictive Relationships between Amyloid 
and Tau Biomarkers in Alzheimer's Disease Progression: An Empirical Model. J Neurosci. 
2019;39(37):7428-7437. doi:10.1523/JNEUROSCI.1056-19.2019 
2. Objective 
Analyzing CSF samples from different age groups to identify biomarkers that undergo 
significant changes during the aging process, such as amyloid-beta (Aβ42), tau protein 
(including its phosphorylated form, p-tau181), and other proteins related to neuroinflammation 
and blood-brain barrier function. 
Establishing a predictive model through the analysis of CSF protein levels and 
classification across different age stages, to enable the early diagnosis of neurodegenerative 
diseases (e.g., Alzheimer's disease). This could allow for the detection of diseases before 
clinical symptoms appear, providing the possibility for early intervention and treatment. 
Investigating the dynamic changes of CSF biomarkers at different stages of disease to 
understand their roles in disease progression, including changes in the early, middle, and late 
stages. This will help to better understand the pathological process and identify potential 

 
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therapeutic windows. 
Creating a database of CSF biomarkers that encompasses different age groups, disease 
states, and treatment responses. This database will provide valuable data resources for future 
research, facilitating the scientific community’s understanding of neurodegenerative diseases 
and the development of new therapies. 
Through the achievement of these objectives, this study aims to advance the development 
of CSF biomarkers in neuroscience research and clinical applications, improving the diagnosis, 
management, and treatment of aging and neurodegenerative diseases. 
3. Method  
Prospective Collection of Cerebrospinal Fluid from Patients Aged 0-90 Years Without 
Neurological Diseases: 
Study Duration: 1 year, starting from the date of ethical approval and ending 1 calendar 
year after the date of ethical approval. 
Inclusion Criteria: 
Patients aged 0-90 years. 
Patients admitted for surgery under spinal anesthesia due to non-neurological 
conditions such as lower limb fractures, hemorrhoids, etc. 
Pediatric patients requiring surgery for tethered cord syndrome. 
Exclusion Criteria: Patients with neurological diseases, including but not limited to 
encephalitis, Parkinson's disease, Alzheimer's disease, hydrocephalus, brain tumors, and 
psychiatric disorders. 
Withdrawal Criteria: Participants may voluntarily withdraw from the study at any time 
for any reason. 
Specific Research Protocol: 
Data Collection: Clinical data will be collected, including the patient's name, gender, 
age, and primary diagnosis. This information will be sourced from the hospital admission 
case system. 
Cerebrospinal Fluid (CSF) Collection: 

 
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During spinal anesthesia, CSF is withdrawn to confirm the correct placement in the 
subarachnoid space before administering the anesthetic, with approximately 1-2 mL of CSF 
retained. 
During surgery for tethered cord syndrome, 2 mL of CSF is collected before opening 
the dura mater. 
The collected CSF is placed in a 0°C insulated container for storage and transportation, 
then centrifuged in the laboratory and stored at -80°C until further analysis. 
After the collection is complete, multi-omics analyses, including proteomics and 
metabolomics, will be conducted. 
Proteomics Analysis: 
Sample Preparation: 
100 μL of CSF is mixed with high-abundance protein depletion resin to enhance 
proteome depth, incubated at room temperature for 20 minutes, and the eluate is collected. 
The sample is concentrated using a 3k molecular weight cutoff protein concentrator at 
12,000 g for 30 minutes and exchanged with lysis buffer (8M urea) for protein denaturation. 
After centrifugation at 12,000 g, less than 50 μL of solution remains in the tube. 
The concentrated sample is reduced with 10 mM tris(2-carboxyethyl)phosphine 
hydrochloride at 32°C for 40 minutes, followed by alkylation with 40 mM iodoacetamide 
at room temperature in the dark for 30 minutes. 
CSF samples are digested in two steps using 2.5 μg of endoproteinase and 2.5 μg of 
trypsin for 12 hours. 
The peptides are acidified with trifluoroacetic acid, desalted using a 2 mg liquid-phase 
ion exchange column, and the eluate is dried using a vacuum concentrator. 
Each batch is labeled with 56 μg of TMT16plex reagent for peptides from each 
sample. After 1-hour incubation at room temperature, TMT labeling is quenched with 

 
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hydroxylamine, and 16 samples from a batch are pooled and fractionated using a C18 
column. 
Fractions are separated into 96 fractions with a gradient from 5% to 35% acetonitrile 
in 10 mM ammonia water at a flow rate of 1 mL/min. The first to seventh, 37th, and 67th 
fractions are pooled, and the remaining fractions are evenly pooled and vacuum dried. 
The dried peptide powder is reconstituted in 2% acetonitrile/0.1% formic acid and 
analyzed by LC-MS/MS. 
Data Analysis: 
MS raw data is searched against the human protein fasta file downloaded from 
UniProtKB14, which contains 20,394 protein entries. 
The parent ion mass tolerance is set to 10 ppm, and the fragment ion mass tolerance 
is set to 0.02 Da. 
Subsequent statistical analysis is performed by comparing the abundance ratios of 
samples within the same batch to the combined sample. 
Bioinformatics software is used to analyze the mass spectrometry data, including 
protein identification, quantification, and differential analysis. 
4. sample capacity 
Participants will be divided into 9 groups based on age, with each group covering a 10-year 
range (1-10 years, 11-20 years, etc.), totaling 9 groups. Each group will include 10-15 
participants, with a planned total enrollment of 100-150 cases. 
5. Data management and confidentiality 
Data Entry and Modification: 
Data entry and management will be handled by Zhu Zhoule and Ying Yuqi. To ensure 
accuracy, two data administrators will independently enter and cross-check the data. 
Database Locking: 
Once the database has been confirmed to be accurate, it will be locked by the principal 
investigator and statistical analysts. All records related to participant identities will be kept 
confidential and will not be disclosed outside the scope allowed by relevant laws and/or 

 
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regulations. 
6. informed consent 
Before the clinical study begins, the researchers must provide detailed information to 
the participants or their legal guardians, including the nature of the study, its purpose, 
potential benefits, and risks. This ensures that participants or their legal guardians fully 
understand the clinical study. The clinical study can only commence after the informed 
consent form has been signed. 
Each patient must provide detailed contact information, including their address and 
phone number. Additionally, the researcher will sign the informed consent form with the 
patient before the surgery and provide the patient with their contact information so that the 
patient can reach the researcher at any time. 
7. Adverse Event Reporting 
This study involves the collection of 1-2 mL of cerebrospinal fluid (CSF) under spinal 
anesthesia. A very small number of patients may experience headaches or discomfort after 
the procedure, which can be alleviated by increasing fluid intake. 
General Adverse Events: Timely measures will be taken to address any adverse events, 
and they will be recorded in the case report form. 
Serious Adverse Events (SAE): Timely measures will be taken to address any SAE. 
The event will be recorded in the case report form, and the researcher will decide whether 
to stop or reduce treatment. The SAE must be reported immediately to the Ethics 
Committee, the drug clinical trial institution, and the sponsor. Additionally, within 24 hours, 
it must be reported to the National and Provincial Food and Drug Administration. The SAE 
must also be reported through the "Internal Adverse Event and Near-Miss No-Fault 
Reporting System." For the detailed process, see the diagram below.