#METABOLOMICS WORKBENCH dmitryleontyev_20240408_074020 DATATRACK_ID:4764 STUDY_ID:ST003165 ANALYSIS_ID:AN005193 PROJECT_ID:PR001969 VERSION 1 CREATED_ON April 9, 2024, 12:26 pm #PROJECT PR:PROJECT_TITLE Spatial Lipidomics Maps Brain Alterations Associated with Mild Traumatic Brain PR:PROJECT_TITLE Injury. PR:PROJECT_SUMMARY Traumatic brain injury (TBI) is a global public health problem with 50-60 PR:PROJECT_SUMMARY million incidents per year, most of which are considered mild (mTBI) and many of PR:PROJECT_SUMMARY these repetitive (rmTBI). Despite their massive implications, the pathologies of PR:PROJECT_SUMMARY mTBI and rmTBI are not fully understood, with a paucity of information on brain PR:PROJECT_SUMMARY lipid dysregulation following mild injury event(s). To gain more insight on mTBI PR:PROJECT_SUMMARY and rmTBI pathology, a non-targeted spatial lipidomics workflow utilizing PR:PROJECT_SUMMARY ultrahigh resolution mass spectrometry imaging was developed to map brain PR:PROJECT_SUMMARY region-specific lipid alterations in rats following injury. Discriminant PR:PROJECT_SUMMARY multivariate models were created for regions of interest including the PR:PROJECT_SUMMARY hippocampus, cortex, and corpus callosum to pinpoint lipid species that PR:PROJECT_SUMMARY differentiated between injured and sham animals. A multivariate model focused on PR:PROJECT_SUMMARY the hippocampus region differentiated injured brain tissues with an area under PR:PROJECT_SUMMARY the curve of 0.994 using only four lipid species. Lipid classes that were PR:PROJECT_SUMMARY consistently discriminant included polyunsaturated fatty acid-containing PR:PROJECT_SUMMARY phosphatidylcholines (PC), lysophosphatidylcholines (LPC), LPC-plasmalogens PR:PROJECT_SUMMARY (LPC-P) and PC potassium adducts. Many of the polyunsaturated fatty PR:PROJECT_SUMMARY acid-containing PC and LPC-P selected have never been previously reported as PR:PROJECT_SUMMARY altered in mTBI. The observed lipid alterations indicate that neuroinflammation PR:PROJECT_SUMMARY and , oxidative stress and disrupted sodium-potassium pumps are important PR:PROJECT_SUMMARY pathologies that could serve to explain cognitive deficits associated with PR:PROJECT_SUMMARY rmTBI. Therapeutics which target or attenuate these pathologies may be PR:PROJECT_SUMMARY beneficial to limit persistent damage following a mild brain injury event. PR:INSTITUTE Georgia Institute of Technology PR:LAST_NAME Leontyev PR:FIRST_NAME Dmitry PR:ADDRESS 311 Ferst Dr NW Atlanta GA 30332 PR:EMAIL dleontyev3@gatech.edu PR:PHONE 301 538 2301 #STUDY ST:STUDY_TITLE Spatial Lipidomics Maps Brain Alterations Associated with Mild Traumatic Brain ST:STUDY_TITLE Injury. ST:STUDY_SUMMARY Traumatic brain injury (TBI) is a global public health problem with 50-60 ST:STUDY_SUMMARY million incidents per year, most of which are considered mild (mTBI) and many of ST:STUDY_SUMMARY these repetitive (rmTBI). Despite their massive implications, the pathologies of ST:STUDY_SUMMARY mTBI and rmTBI are not fully understood, with a paucity of information on brain ST:STUDY_SUMMARY lipid dysregulation following mild injury event(s). To gain more insight on mTBI ST:STUDY_SUMMARY and rmTBI pathology, a non-targeted spatial lipidomics workflow utilizing ST:STUDY_SUMMARY ultrahigh resolution mass spectrometry imaging was developed to map brain ST:STUDY_SUMMARY region-specific lipid alterations in rats following injury. Discriminant ST:STUDY_SUMMARY multivariate models were created for regions of interest including the ST:STUDY_SUMMARY hippocampus, cortex, and corpus callosum to pinpoint lipid species that ST:STUDY_SUMMARY differentiated between injured and sham animals. A multivariate model focused on ST:STUDY_SUMMARY the hippocampus region differentiated injured brain tissues with an area under ST:STUDY_SUMMARY the curve of 0.994 using only four lipid species. Lipid classes that were ST:STUDY_SUMMARY consistently discriminant included polyunsaturated fatty acid-containing ST:STUDY_SUMMARY phosphatidylcholines (PC), lysophosphatidylcholines (LPC), LPC-plasmalogens ST:STUDY_SUMMARY (LPC-P) and PC potassium adducts. Many of the polyunsaturated fatty ST:STUDY_SUMMARY acid-containing PC and LPC-P selected have never been previously reported as ST:STUDY_SUMMARY altered in mTBI. The observed lipid alterations indicate that neuroinflammation ST:STUDY_SUMMARY and , oxidative stress and disrupted sodium-potassium pumps are important ST:STUDY_SUMMARY pathologies that could serve to explain cognitive deficits associated with ST:STUDY_SUMMARY rmTBI. Therapeutics which target or attenuate these pathologies may be ST:STUDY_SUMMARY beneficial to limit persistent damage following a mild brain injury event. ST:INSTITUTE Georgia Institute of Technology ST:LAST_NAME Leontyev ST:FIRST_NAME Dmitry ST:ADDRESS 311 Ferst Dr NW Atlanta GA 30332 ST:EMAIL dleontyev3@gatech.edu ST:PHONE 301 538 2301 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 SU:GENDER Male #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS 1 1 Condition:SHAM | Sample source:Brain RAW_FILE_NAME(Raw file name)=Rat 1 Set 2 Slide 1.imzML SUBJECT_SAMPLE_FACTORS 2 2 Condition:TBI | Sample source:Brain RAW_FILE_NAME(Raw file name)=Rat 2 Set 2 Slide 5.imzML SUBJECT_SAMPLE_FACTORS 3 3 Condition:TBI | Sample source:Brain RAW_FILE_NAME(Raw file name)=Rat3left.imzML SUBJECT_SAMPLE_FACTORS 4 4 Condition:SHAM | Sample source:Brain RAW_FILE_NAME(Raw file name)=Rat4left.imzML SUBJECT_SAMPLE_FACTORS 5 5 Condition:TBI | Sample source:Brain RAW_FILE_NAME(Raw file name)=Rat5left.imzML SUBJECT_SAMPLE_FACTORS 6 6 Condition:SHAM | Sample source:Brain RAW_FILE_NAME(Raw file name)=Rat6right.imzML SUBJECT_SAMPLE_FACTORS 8 8 Condition:SHAM | Sample source:Brain RAW_FILE_NAME(Raw file name)=Rat8right.imzML SUBJECT_SAMPLE_FACTORS 9 9 Condition:SHAM | Sample source:Brain RAW_FILE_NAME(Raw file name)=Rat9left.imzML SUBJECT_SAMPLE_FACTORS 10 10 Condition:TBI | Sample source:Brain RAW_FILE_NAME(Raw file name)=Rat10left.imzML SUBJECT_SAMPLE_FACTORS 11 11 Condition:TBI | Sample source:Brain RAW_FILE_NAME(Raw file name)=Rat11left.imzML SUBJECT_SAMPLE_FACTORS 12 12 Condition:TBI | Sample source:Brain RAW_FILE_NAME(Raw file name)=Rat12left.imzML #COLLECTION CO:COLLECTION_SUMMARY A total of twenty images from eleven rats were used for multivariate image CO:COLLECTION_SUMMARY analysis with eight of those sections being from sham animals and twelve from CO:COLLECTION_SUMMARY injured animals. CO:SAMPLE_TYPE Brain #TREATMENT TR:TREATMENT_SUMMARY Prior to injury, rats were anesthetized and maintained with 2-3% isoflurane. TR:TREATMENT_SUMMARY Rats were then placed on 1-inch-thick ethylene-vinyl acetate foam TR:TREATMENT_SUMMARY (McMaster-Carr, Elmhurst, IL, USA). rmTBI was induced by subjecting rats to TR:TREATMENT_SUMMARY three closed head impacts (2 min interval, 5 m s-1 velocity, 5 mm, 2 mm, and 2 TR:TREATMENT_SUMMARY mm head displacement) to the dorsal head surface using a CCI pneumatic injury TR:TREATMENT_SUMMARY device (Pittsburgh Precision Instruments, Pittsburgh, PA, USA). This device was TR:TREATMENT_SUMMARY equipped with a 1-cm diameter silicone stopper (Renovators Supply Manufacturing, TR:TREATMENT_SUMMARY Erving, MA, USA) attached to the piston tip. The sham group underwent identical TR:TREATMENT_SUMMARY procedures as the injured group, except for the impacts. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Sagittal 12-µm sections collected serially from right brain hemispheres using a SP:SAMPLEPREP_SUMMARY cryostat (Thermo Shandon NX70 Cryostar, Waltham, MA) were mounted onto SP:SAMPLEPREP_SUMMARY indium-tin-oxide (ITO) slides (Delta Technologies, Loveland, CO) and stored at SP:SAMPLEPREP_SUMMARY -80°C until MALDI MSI. Prior to imaging, slides were placed in a desiccator for SP:SAMPLEPREP_SUMMARY 15 minutes and sprayed with 8 passes of a 5 mg mL-1 DAN solution in 90% SP:SAMPLEPREP_SUMMARY acetonitrile/10% water using an HTX TM-Sprayer (HTX Technologies, Chapel Hill, SP:SAMPLEPREP_SUMMARY NC) at 30°C, 0.1 mL min-1 flow rate, 1200 mm min-1 velocity, 2.5 mm tracking SP:SAMPLEPREP_SUMMARY speed, 10 psi, 2 L min-1 gas flow rate, 0 second drying time and 40 mm nozzle SP:SAMPLEPREP_SUMMARY height. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE None (Direct infusion) CH:INSTRUMENT_NAME Bruker solariX 12T CH:COLUMN_NAME none CH:SOLVENT_A N/A CH:SOLVENT_B N/A CH:FLOW_GRADIENT N/A CH:FLOW_RATE N/A CH:COLUMN_TEMPERATURE N/A #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker Solarix FT-ICR-MS MS:INSTRUMENT_TYPE FT-ICR MS:MS_TYPE MALDI MS:ION_MODE POSITIVE MS:MS_COMMENTS Provided are .imzML/.ibd files that are root mean square normalized when MS:MS_COMMENTS exported from SCiLS Lab. I am providing 1 section from each brain. MALDI imaging MS:MS_COMMENTS data were collected on a solariX 12T FTICR mass spectrometer (Bruker Daltonics, MS:MS_COMMENTS Bremen, Germany) in positive ion mode in the m/z 147-1500 range using 2M MS:MS_COMMENTS transients (~300,000 mass resolution at m/z 314). A 50 µm raster spacing in the MS:MS_COMMENTS x and y directions was used. The laser was set to 100 shots, small focus, 12 % MS:MS_COMMENTS power and 1000 Hz. Real time calibration with a lock mass of m/z 314.152598 from MS:MS_COMMENTS the DAN dimer and m/z 760.585082 from PC(34:1) was used to achieve optimal mass MS:MS_COMMENTS accuracy. Two sections from each of the five sham and six injured brains were MS:MS_COMMENTS examined by FTICR MSI. Serial sections were placed on the same ITO slide at the MS:MS_COMMENTS time of the MSI experiment. Among the sections examined, two replicate images MS:MS_COMMENTS were eliminated from the dataset due to abnormally low ion abundances. A total MS:MS_COMMENTS of twenty images from eleven rats were used for multivariate image analysis with MS:MS_COMMENTS eight of those sections being from sham animals and twelve from injured animals. MS:MS_COMMENTS MS images were uploaded to, and analyzed in SCiLS Lab Version 2022b Pro (Bruker MS:MS_COMMENTS Daltonics, Bremen, Germany). Regarding pre-processing options, no baseline MS:MS_COMMENTS correction or other notable options were used. For segmentation purposes, a MS:MS_COMMENTS feature list was created with the sliding window tool using the average mass MS:MS_COMMENTS spectrum from all brain sections and the lowest possible intensity threshold. MS:MS_COMMENTS This yielded a large peak list with a ± 3 ppm window for each ion. This feature MS:MS_COMMENTS list was used to computationally segment the brain images into molecularly MS:MS_COMMENTS similar regions of interest (ROI). The parameters used for segmentation were the MS:MS_COMMENTS preliminary feature list, root mean square normalization, strong denoising, MS:MS_COMMENTS bisecting k-means and the Manhattan distance metric. Segmentation was performed MS:MS_COMMENTS on individual brain sections or regions. In a few cases, some ROI were not MS:MS_COMMENTS correctly picked out by the automated segmentation approach alone and were thus MS:MS_COMMENTS manually outlined following specific lipid distributions that helped delineate MS:MS_COMMENTS the ROI borders. For each ROI, feature lists were first created in SCiLS Lab MS:MS_COMMENTS with a ± 5 ppm feature tolerance. Receiver operating characteristic (ROC) MS:MS_COMMENTS analysis was then conducted on these ROI-specific feature lists using the mean MS:MS_COMMENTS spectra. All ions with an area under the curve above 0.7 (i.e., those with MS:MS_COMMENTS abundances larger in control brains), or those with an area under the curve MS:MS_COMMENTS (AUC) below 0.3 (more abundant in injured animals) were chosen. The remaining MS:MS_COMMENTS m/z values were filtered out from the input feature list. For ROI involving the MS:MS_COMMENTS gray matter, the white matter, and the corpus callosum, AUC cutoff values were MS:MS_COMMENTS set to a stricter cutoff of 0.8 and 0.2, as the corresponding input feature MS:MS_COMMENTS lists contained a large abundance of ions. Extracted ion images for m/z values MS:MS_COMMENTS in the resulting feature lists were inspected to remove any species originating MS:MS_COMMENTS from the MALDI matrix, the embedding mixture, or ions with poor signal-to-noise MS:MS_COMMENTS ratios. The spectral profile for each feature was inspected to ensure that the MS:MS_COMMENTS interval chosen by SCiLS software was correctly aligned with the apex of each MS:MS_COMMENTS peak, and the feature list interval tolerance then lowered to ± 3 ppm. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS intensity MS_METABOLITE_DATA_START Samples 1 1 10 10 11 11 12 12 2 2 3 3 4 4 5 5 6 8 9 9 Factors Condition:SHAM | Sample source:Brain Condition:SHAM | Sample source:Brain Condition:TBI | Sample source:Brain Condition:TBI | Sample source:Brain Condition:TBI | Sample source:Brain Condition:TBI | Sample source:Brain Condition:TBI | Sample source:Brain Condition:TBI | Sample source:Brain Condition:TBI | Sample source:Brain Condition:TBI | Sample source:Brain Condition:TBI | Sample source:Brain Condition:TBI | Sample source:Brain Condition:SHAM | Sample source:Brain Condition:SHAM | Sample source:Brain Condition:TBI | Sample source:Brain Condition:TBI | Sample source:Brain Condition:SHAM | Sample source:Brain Condition:SHAM | Sample source:Brain Condition:SHAM | Sample source:Brain Condition:SHAM | Sample source:Brain LPC 16:0 2989.29224 4087.45239 5826.10449 5903.63818 5704.38818 5300.06152 5829.64209 5546.41016 4842.75781 4889.28467 5336.34766 5375.72412 5299.96826 5346.26953 5930.28711 5675.55518 3936.75073 5003.56445 4086.1145 4609.97266 PC 36:1 61051.6016 59506.1992 66412.8672 68135.4844 56602.1875 56230.207 54689.125 56435 52392.1914 53794.2617 59749.7617 60700.9648 59724.293 61201.2031 57178.6992 57557.4531 65621.8828 61754.9219 61898.8906 61748.6172 PC 40:6 13591.0986 14495.4229 13198.8525 13184.4277 13807.9521 13164.1465 13123.1543 13287.8262 12499.3691 12805.5195 12589.8779 12423.1445 14306.4414 14228.4189 12258.7773 12311.4014 15025.4922 14355.9365 13261.4863 13248.8447 SM 42:2 [M+K] 200.281311 286.526398 95.2031784 156.759216 143.861053 96.2549667 62.1748543 72.0863876 269.23172 318.106873 93.7046738 60.7599983 679.97998 398.592896 363.243988 144.123749 435.11026 103.632683 406.518829 421.022919 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name LIPIDMAPS ID PubChemID LPC 16:0 LMGP01050074 15061532 PC 36:1 LMGP01011376 52922234 PC 40:6 LMGP01010821 24778876 SM 42:2 [M+K] LMSP03010007 44260126 METABOLITES_END #END