Summary of Study ST003767

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002350. The data can be accessed directly via it's Project DOI: 10.21228/M8VV7X This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

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Study ID	ST003767
Study Title	N-acetylneuraminic Acid may play Links Secondary Neurodegeneration and Cognitive Impairment in Cortical Photothrombotic Stroke Mouse Model
Study Summary	ABSTRACT Background: Post-stroke cognitive impairment (PSCI) is a major sequelae of ischemic stroke (IS), but the associated mechanisms remain unclear. Recent studies have shown that cortical stroke can cause distal brain disturbances, leading to cognitive decline. This study aims to investigate the neuropathological and metabolic changes over time in the remote brain region associated with cognitive impairment after cortical stroke. Methods: We assessed cognitive function in photothrombotic (PT) mice for 84 days and identified distal brain regions with secondary neurodegeneration (SND) using voxel-based morphometry (VBM). Integrated untargeted and targeted metabolomics method was established to comprehensively analyze the molecular mechanism of SND in this brain region and screen the potential predictive biomarker of PSCI. Furthermore, the mechanism of the biomarker with PSCI and SND was studied in vitro. Results: The results show that recent memory impairment, remote dysfunction, and anxiety persisted for 84 days in PT mice. The hippocampus of cognitively impaired PT mice developed SND and metabolic disorders, particularly oxidative stress, lipid peroxidation, and inflammation. N-acetylneuramic acid (Neu5Ac) was screened in the hippocampus of mice, serum of mice and 154 IS patients. Neu5Ac promotes oxidative stress and inflammation in microglia. Conclusions: Our results suggest that hippocampal SND is closely related to cognitive impairment in PT mice, while oxidative stress and inflammation are important factors in hippocampal SND. Neu5Ac may aggravate PSCI by causing hippocampal SND through inducting of microglia-driven oxidative stress and inflammation, and it may thus be a potential predictive biomarker of PSCI
Institute	Wenzhou Medical University
Last Name	Zhou
First Name	Yiyang
Address	University Town, Chashan, Wenzhou, Zhejiang, 325035 P.R China
Email	zhouyiyang@wmu.edu.cn
Phone	13806831161
Submit Date	2025-02-21
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2025-06-24
Release Version	1

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Project:

Project ID:	PR002350
Project DOI:	doi: 10.21228/M8VV7X
Project Title:	N-acetylneuraminic Acid may play Links Secondary Neurodegeneration and Cognitive Impairment in Cortical Photothrombotic Stroke Mouse Model
Project Summary:	ABSTRACT Background: Post-stroke cognitive impairment (PSCI) is a major sequelae of ischemic stroke (IS), but the associated mechanisms remain unclear. Recent studies have shown that cortical stroke can cause distal brain disturbances, leading to cognitive decline. This study aims to investigate the neuropathological and metabolic changes over time in the remote brain region associated with cognitive impairment after cortical stroke. Methods: We assessed cognitive function in photothrombotic (PT) mice for 84 days and identified distal brain regions with secondary neurodegeneration (SND) using voxel-based morphometry (VBM). Integrated untargeted and targeted metabolomics method was established to comprehensively analyze the molecular mechanism of SND in this brain region and screen the potential predictive biomarker of PSCI. Furthermore, the mechanism of the biomarker with PSCI and SND was studied in vitro. Results: The results show that recent memory impairment, remote dysfunction, and anxiety persisted for 84 days in PT mice. The hippocampus of cognitively impaired PT mice developed SND and metabolic disorders, particularly oxidative stress, lipid peroxidation, and inflammation. N-acetylneuramic acid (Neu5Ac) was screened in the hippocampus of mice, serum of mice and 154 IS patients. Neu5Ac promotes oxidative stress and inflammation in microglia. Conclusions: Our results suggest that hippocampal SND is closely related to cognitive impairment in PT mice, while oxidative stress and inflammation are important factors in hippocampal SND. Neu5Ac may aggravate PSCI by causing hippocampal SND through inducting of microglia-driven oxidative stress and inflammation, and it may thus be a potential predictive biomarker of PSCI.
Institute:	Wenzhou Medical University
Last Name:	Zhou
First Name:	Yiyang
Address:	University Town, Chashan, Wenzhou, Zhejiang, 325035 P.R China
Email:	zhouyiyang@wmu.edu.cn
Phone:	+8613806831161

Subject:

Subject ID:	SU003900
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample_type
SA409532	C17	C1
SA409533	C12	C1
SA409534	C19	C1
SA409535	C11	C1
SA409536	C16	C1
SA409537	C13	C1
SA409538	C15	C1
SA409539	C14	C1
SA409540	C21	C2
SA409541	C23	C2
SA409542	C24	C2
SA409543	C25	C2
SA409544	C26	C2
SA409545	C27	C2
SA409546	C28	C2
SA409547	C22	C2
SA409548	C37	C3
SA409549	C39	C3
SA409550	C38	C3
SA409551	C35	C3
SA409552	C36	C3
SA409553	C32	C3
SA409554	C31	C3
SA409555	C33	C3
SA409556	QC-1	Control
SA409557	QC-6	Control
SA409558	QC-5	Control
SA409559	QC-4	Control
SA409560	QC-3	Control
SA409561	QC-2	Control
SA409562	M17	M1
SA409563	M14	M1
SA409564	M11	M1
SA409565	M12	M1
SA409566	M13	M1
SA409567	M15	M1
SA409568	M16	M1
SA409569	M18	M1
SA409570	M22	M2
SA409571	M21	M2
SA409572	M28	M2
SA409573	M29	M2
SA409574	M26	M2
SA409575	M25	M2
SA409576	M24	M2
SA409577	M23	M2
SA409578	M32	M3
SA409579	M33	M3
SA409580	M31	M3
SA409581	M35	M3
SA409582	M36	M3
SA409583	M38	M3
SA409584	M39	M3
SA409585	M34	M3

Showing results 1 to 54 of 54

Showing results 1 to 54 of 54

Collection:

Collection ID:	CO003893
Collection Summary:	After completing behavioral and imaging experiments, the mice were decapitated under isoflurane anesthesia and their brains were immediately extracted.Therefore, the hippocampus was extracted from the brain tissue based on its anatomical features for further analysis and the tissue was either rapidly frozen in liquid nitrogen, stored at -80°C for metabolomics analysis.
Sample Type:	Hippocampus

Treatment:

Treatment ID:	TR003909
Treatment Summary:	After one week of acclimatization, 8-week-old C57BL/6J mice were randomly divided into a sham surgery group (Sham) and a photothrombotic surgery group (Photothrombotic surgery, PT). We utilized a PT stroke model targeting the frontal cortex of mice, which has been demonstrated to be suitable for studying post-stroke dementia. Mice in the PT group were anesthetized with 2% isoflurane and maintained under general anesthesia throughout the procedure. Rose Bengal (200 µL, 10 mg/mL in sterile saline solution, Sigma-Aldrich, USA) was intraperitoneally injected and circulated for 8 minutes. Subsequently, a cold light source with a diameter of 4.5 mm was used to irradiate the exposed skull for 15 minutes, positioned 3.5 mm to the left of the bregma, targeting the left frontal lobe. The Sham group underwent the same surgical procedure but was administered 200 µL of sterile saline (0.9% NaCl, Pfizer, Australia) instead of Rose Bengal. Both the Sham and PT groups were then further divided based on the time post-stroke into T1 (C1 n=11, M1 n=11), T2 (C2 n=11, M2 n=11), and T3 (C3 n=11, M3 n=11) groups, where C1, C2, and C3 represent the Sham groups at 14, 32, and 84 days post-stroke, respectively, and M1, M2, and M3 represent the PT groups at 14, 32, and 84 days post-stroke, respectively.

Treatment ID:

TR003909

Treatment Summary:

After one week of acclimatization, 8-week-old C57BL/6J mice were randomly divided into a sham surgery group (Sham) and a photothrombotic surgery group (Photothrombotic surgery, PT). We utilized a PT stroke model targeting the frontal cortex of mice, which has been demonstrated to be suitable for studying post-stroke dementia. Mice in the PT group were anesthetized with 2% isoflurane and maintained under general anesthesia throughout the procedure. Rose Bengal (200 µL, 10 mg/mL in sterile saline solution, Sigma-Aldrich, USA) was intraperitoneally injected and circulated for 8 minutes. Subsequently, a cold light source with a diameter of 4.5 mm was used to irradiate the exposed skull for 15 minutes, positioned 3.5 mm to the left of the bregma, targeting the left frontal lobe. The Sham group underwent the same surgical procedure but was administered 200 µL of sterile saline (0.9% NaCl, Pfizer, Australia) instead of Rose Bengal. Both the Sham and PT groups were then further divided based on the time post-stroke into T1 (C1 n=11, M1 n=11), T2 (C2 n=11, M2 n=11), and T3 (C3 n=11, M3 n=11) groups, where C1, C2, and C3 represent the Sham groups at 14, 32, and 84 days post-stroke, respectively, and M1, M2, and M3 represent the PT groups at 14, 32, and 84 days post-stroke, respectively.

Sample Preparation:

Sampleprep ID:	SP003906
Sampleprep Summary:	Take 10 mg of mouse hippocampus in a 1.5 mL EP tube, add 400 µL of cold acetonitrile:methanol (v:v = 1:1) to 200 µL of plasma to precipitate proteins, mix for 60 seconds, and then centrifuge to collect the supernatant. Sequentially add methanol:water:dichloromethane (v:v:v = 1:1:2), three mass spectrometry-grade extraction solvents, homogenize at 60 Hz for 2 minutes, and let stand at 4°C for 30 minutes. Centrifuge at 4°C, 15000 rpm for 15 minutes. The liquid separates into two layers: proteins and tissue precipitate in the middle, the upper layer is a methanol-water solution mainly containing polar small molecules, and the lower layer is a dichloromethane solution mainly composed of lipid-soluble small molecules. Take the upper layer solution, dry it under N2, and reconstitute it with 200 µL of 50% acetonitrile aqueous solution containing an internal standard (2-chlorophenylalanine). Centrifuge at 4°C, 15000 rpm for 15 minutes, then draw 60 µL and inject it into a liquid phase vial with a glass liner for analysis. Take the lower layer solution, dry it under N2, and reconstitute it with 100 µL of chloroform (dichloromethane):methanol = 1:1 solution containing an internal standard (2-chlorophenylalanine). Centrifuge at 4°C, 15000 rpm for 15 minutes, then draw 100 µL and inject it into a liquid phase vial with a glass liner for analysis. Simultaneously, take 10 µL of all supernatant to prepare Quality Control (QC) samples to monitor the stability of the instrument acquisition method.

Sampleprep ID:

SP003906

Sampleprep Summary:

Take 10 mg of mouse hippocampus in a 1.5 mL EP tube, add 400 µL of cold acetonitrile:methanol (v:v = 1:1) to 200 µL of plasma to precipitate proteins, mix for 60 seconds, and then centrifuge to collect the supernatant. Sequentially add methanol:water:dichloromethane (v:v:v = 1:1:2), three mass spectrometry-grade extraction solvents, homogenize at 60 Hz for 2 minutes, and let stand at 4°C for 30 minutes. Centrifuge at 4°C, 15000 rpm for 15 minutes. The liquid separates into two layers: proteins and tissue precipitate in the middle, the upper layer is a methanol-water solution mainly containing polar small molecules, and the lower layer is a dichloromethane solution mainly composed of lipid-soluble small molecules. Take the upper layer solution, dry it under N2, and reconstitute it with 200 µL of 50% acetonitrile aqueous solution containing an internal standard (2-chlorophenylalanine). Centrifuge at 4°C, 15000 rpm for 15 minutes, then draw 60 µL and inject it into a liquid phase vial with a glass liner for analysis. Take the lower layer solution, dry it under N2, and reconstitute it with 100 µL of chloroform (dichloromethane):methanol = 1:1 solution containing an internal standard (2-chlorophenylalanine). Centrifuge at 4°C, 15000 rpm for 15 minutes, then draw 100 µL and inject it into a liquid phase vial with a glass liner for analysis. Simultaneously, take 10 µL of all supernatant to prepare Quality Control (QC) samples to monitor the stability of the instrument acquisition method.

Combined analysis:

Analysis ID	AN006183	AN006184
Chromatography ID	CH004694	CH004694
MS ID	MS005887	MS005888
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Waters Acquity	Waters Acquity
Column	Phenomenex Kinetex C18 (100 x 2.1 mm, 2.6 µm)	Phenomenex Kinetex C18 (100 x 2.1 mm, 2.6 µm)
MS Type	ESI	ESI
MS instrument type	Triple TOF	Triple TOF
MS instrument name	ABI Sciex 6600 TripleTOF	ABI Sciex 6600 TripleTOF
Ion Mode	POSITIVE	NEGATIVE
Units	Peak area	Peak area

Chromatography:

Chromatography ID:	CH004694
Instrument Name:	Waters Acquity
Column Name:	Phenomenex Kinetex C18 (100 x 2.1 mm, 2.6 µm)
Column Temperature:	35°C
Flow Gradient:	0-0.5 min 98% B；0.5-13 min，98-40% B；13-3.1 min，40-98% B；13.1-18 min，98-98%
Flow Rate:	0.3 mL/min
Solvent A:	100% Water; 5 mM ammonium acetate; 0.1% formic acid
Solvent B:	100% Acetonitrile
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005887
Analysis ID:	AN006183
Instrument Name:	ABI Sciex 6600 TripleTOF
Instrument Type:	Triple TOF
MS Type:	ESI
MS Comments:	The electrospray ionization (ESI) positive ion mode was employed, with a mass detection range of 80-1000 m/z, and the collision energy for secondary fragments was set at 40±15 V. After data acquisition, the raw data were preprocessed using Markerview software. The main parameters included: peak extraction time of 0.5-15 minutes, retention time deviation of 0.1 minutes, mass-to-charge ratio deviation of 10 ppm, and peak intensity >500. Metabolites with an overall detection rate of less than 80% were excluded from the analysis. An Excel spreadsheet containing m/z, retention time (RT), and peak intensity was exported. Prior to the next step of analysis, peaks with intensities below 500 were excluded. The coefficient of variation (CV: standard deviation/mean × 100%) was calculated, and peaks with CV% >30% were identified as high-variability peaks and thus also excluded. Multivariate statistical analysis of the data was performed using SMICA-P software (v14.0, Umetrics, Umea, Sweden), primarily including principal component analysis (PCA). PCA is an unsupervised model used to examine changes in the metabolic patterns of the mouse hippocampus. Significant differential metabolites were screened through univariate statistical analysis, and volcano plots were generated to represent these findings. Metabolite identification was primarily based on accurate m/z values and MS/MS characteristic fragments, using software such as MS-DIAL (Mass spectrometry-data independent analysis software), MetDNA2 (http://metdna.zhulab.cn/), and One-MAP (http://www.5omics.com/). These identified metabolites were further validated using HMDB (https://hmdb.ca/). Pathway enrichment analysis was conducted via Metaboanalyst (https://www.metaboanalyst.ca/).
Ion Mode:	POSITIVE

MS ID:	MS005888
Analysis ID:	AN006184
Instrument Name:	ABI Sciex 6600 TripleTOF
Instrument Type:	Triple TOF
MS Type:	ESI
MS Comments:	The electrospray ionization (ESI) negtive ion mode was employed, with a mass detection range of 80-1000 m/z, and the collision energy for secondary fragments was set at -40±15 V. After data acquisition, the raw data were preprocessed using Markerview software. The main parameters included: peak extraction time of 0.5-15 minutes, retention time deviation of 0.1 minutes, mass-to-charge ratio deviation of 10 ppm, and peak intensity >500. Metabolites with an overall detection rate of less than 80% were excluded from the analysis. An Excel spreadsheet containing m/z, retention time (RT), and peak intensity was exported. Prior to the next step of analysis, peaks with intensities below 500 were excluded. The coefficient of variation (CV: standard deviation/mean × 100%) was calculated, and peaks with CV% >30% were identified as high-variability peaks and thus also excluded. Multivariate statistical analysis of the data was performed using SMICA-P software (v14.0, Umetrics, Umea, Sweden), primarily including principal component analysis (PCA). PCA is an unsupervised model used to examine changes in the metabolic patterns of the mouse hippocampus. Significant differential metabolites were screened through univariate statistical analysis, and volcano plots were generated to represent these findings. Metabolite identification was primarily based on accurate m/z values and MS/MS characteristic fragments, using software such as MS-DIAL (Mass spectrometry-data independent analysis software), MetDNA2 (http://metdna.zhulab.cn/), and One-MAP (http://www.5omics.com/). These identified metabolites were further validated using HMDB (https://hmdb.ca/). Pathway enrichment analysis was conducted via Metaboanalyst (https://www.metaboanalyst.ca/).
Ion Mode:	NEGATIVE