#METABOLOMICS WORKBENCH apulliam3_20240216_053758 DATATRACK_ID:4649 STUDY_ID:ST003093 ANALYSIS_ID:AN005062 PROJECT_ID:PR001922 VERSION 1 CREATED_ON February 20, 2024, 11:47 am #PROJECT PR:PROJECT_TITLE Integrative Analysis of Cytokine and Lipidomics Datasets Fol-lowing Mild PR:PROJECT_TITLE Traumatic Brain Injury in the Rat PR:PROJECT_SUMMARY Traumatic brain injury (TBI) is a significant source of disability in the United PR:PROJECT_SUMMARY States and around the world and may lead to long-lasting cognitive deficits and PR:PROJECT_SUMMARY decreased quality of life for patients across injury severities. Following the PR:PROJECT_SUMMARY primary injury phase, TBI is characterized by com-plex secondary cascades that PR:PROJECT_SUMMARY involve altered homeostasis and metabolism, faulty signaling, PR:PROJECT_SUMMARY neu-roinflammation, and lipid dysfunction. The objectives of the present study PR:PROJECT_SUMMARY were to (1) assess po-tential correlations between lipidome and cytokine changes PR:PROJECT_SUMMARY after closed-head mild TBI (mTBI), and (2) examine reproducibility of our acute PR:PROJECT_SUMMARY lipidomic profiles following TBI. Cortices from 54 Sprague Dawley male and PR:PROJECT_SUMMARY female rats were analyzed by ultra-high-performance liquid chromatography mass PR:PROJECT_SUMMARY spectrometry (LC-MS) in both positive and negative ionization modes and PR:PROJECT_SUMMARY multiplex cytokine analysis after single (smTBI) or repetitive (rmTBI) PR:PROJECT_SUMMARY closed-head impacts, or sham conditions. Tissue age was a variable, given that PR:PROJECT_SUMMARY two cohorts (n= 26 and n=28) were initially run a year-and-a-half apart, PR:PROJECT_SUMMARY creating inter-batch variations. We annotated the lipidome datasets using an PR:PROJECT_SUMMARY in-house data dictionary based on exact masses of precursor and fragment ions PR:PROJECT_SUMMARY and removed features with statis-tically significant differences between sham PR:PROJECT_SUMMARY control batches. Our results indicate that lipids with high fold change between PR:PROJECT_SUMMARY injury groups moderately correlate with the cytokines eotaxin, IP-10, and TNF-a. PR:PROJECT_SUMMARY Additionally, we show a significant decrease of the pro-inflammatory markers, PR:PROJECT_SUMMARY IL-1b and IP-10, TNF-a, and RANTES in the rmTBI samples relative to sham PR:PROJECT_SUMMARY control. We discuss the major challenges in correlating high dimensional PR:PROJECT_SUMMARY lipidomic data with functional cytokine profiles and the implications for PR:PROJECT_SUMMARY understanding the biological significance of two related but disparate analysis PR:PROJECT_SUMMARY modes in the study of TBI, an inherently heterogeneous neurological disorder. PR:INSTITUTE Georgia Institute of Technology PR:DEPARTMENT The Wallace H. Coulter Department of Biomedical Engineering PR:LABORATORY Michelle LaPlaca PR:LAST_NAME Pulliam PR:FIRST_NAME Alexis PR:ADDRESS 313 Ferst Dr. NW, Atlanta, GA, 30332 PR:EMAIL apulliam3@gatech.edu PR:PHONE 404.385.0629 #STUDY ST:STUDY_TITLE Integrative Analysis of Cytokine and Lipidomics Datasets Following Mild ST:STUDY_TITLE Traumatic Brain Injury in the Rat ST:STUDY_SUMMARY Traumatic brain injury (TBI) is a significant source of disability in the United ST:STUDY_SUMMARY States and around the world and may lead to long-lasting cognitive deficits and ST:STUDY_SUMMARY decreased quality of life for patients across injury severities. Following the ST:STUDY_SUMMARY primary injury phase, TBI is characterized by com-plex secondary cascades that ST:STUDY_SUMMARY involve altered homeostasis and metabolism, faulty signaling, ST:STUDY_SUMMARY neu-roinflammation, and lipid dysfunction. The objectives of the present study ST:STUDY_SUMMARY were to (1) assess po-tential correlations between lipidome and cytokine changes ST:STUDY_SUMMARY after closed-head mild TBI (mTBI), and (2) examine reproducibility of our acute ST:STUDY_SUMMARY lipidomic profiles following TBI. Cortices from 54 Sprague Dawley male and ST:STUDY_SUMMARY female rats were analyzed by ultra-high-performance liquid chromatography mass ST:STUDY_SUMMARY spectrometry (LC-MS) in both positive and negative ionization modes and ST:STUDY_SUMMARY multiplex cytokine analysis after single (smTBI) or repetitive (rmTBI) ST:STUDY_SUMMARY closed-head impacts, or sham conditions. Tissue age was a variable, given that ST:STUDY_SUMMARY two cohorts (n= 26 and n=28) were initially run a year-and-a-half apart, ST:STUDY_SUMMARY creating inter-batch variations. We annotated the lipidome datasets using an ST:STUDY_SUMMARY in-house data dictionary based on exact masses of precursor and fragment ions ST:STUDY_SUMMARY and removed features with statis-tically significant differences between sham ST:STUDY_SUMMARY control batches. Our results indicate that lipids with high fold change between ST:STUDY_SUMMARY injury groups moderately correlate with the cytokines eotaxin, IP-10, and TNF-a. ST:STUDY_SUMMARY Additionally, we show a significant decrease of the pro-inflammatory markers, ST:STUDY_SUMMARY IL-1b and IP-10, TNF-a, and RANTES in the rmTBI samples relative to sham ST:STUDY_SUMMARY control. We discuss the major challenges in correlating high dimensional ST:STUDY_SUMMARY lipidomic data with functional cytokine profiles and the implications for ST:STUDY_SUMMARY understanding the biological significance of two related but disparate analysis ST:STUDY_SUMMARY modes in the study of TBI, an inherently heterogeneous neurological disorder. ST:INSTITUTE Georgia Institute of Technology ST:DEPARTMENT The Wallace H. Coulter Department of Biomedical Engineering ST:LABORATORY Michelle LaPlaca ST:LAST_NAME Pulliam ST:FIRST_NAME Alexis ST:ADDRESS 313 Ferst Dr. NW, Atlanta, GA, 30332 ST:EMAIL apulliam3@gatech.edu ST:PHONE 404.385.0629 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 54 ST:NUM_MALES 27 ST:NUM_FEMALES 27 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 SU:WEIGHT_OR_WEIGHT_RANGE 400g SU:GENDER Male and female SU:ANIMAL_ANIMAL_SUPPLIER Charles River SU:ANIMAL_HOUSING Double Housed SU:ANIMAL_LIGHT_CYCLE Reverse Light Cycle SU:SPECIES_GROUP Sprague Dawley #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 - 01_013 Sample source:Norm. Area 01_13.raw (F32) | Injury severity:1X | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_13.raw SUBJECT_SAMPLE_FACTORS - 01_017 Sample source:Norm. Area 01_17.raw (F50) | Injury severity:1X | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_17.raw SUBJECT_SAMPLE_FACTORS - 01_022 Sample source:Norm. Area 01_22.raw (F46) | Injury severity:1X | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_22.raw SUBJECT_SAMPLE_FACTORS - 01_024 Sample source:Norm. Area 01_24.raw (F29) | Injury severity:1X | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_24.raw SUBJECT_SAMPLE_FACTORS - 01_027 Sample source:Norm. Area 01_27.raw (F59) | Injury severity:1X | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_27.raw SUBJECT_SAMPLE_FACTORS - 01_010 Sample source:Norm. Area 01_10.raw (F77) | Injury severity:1X | Sex:M | Batches:Batch 1 RAW_FILE_NAME=01_10.raw SUBJECT_SAMPLE_FACTORS - 01_006 Sample source:Norm. Area 01_6.raw (F79) | Injury severity:1X | Sex:M | Batches:Batch 1 RAW_FILE_NAME=01_6.raw SUBJECT_SAMPLE_FACTORS - 01_008 Sample source:Norm. Area 01_8.raw (F76) | Injury severity:1X | Sex:M | Batches:Batch 1 RAW_FILE_NAME=01_8.raw SUBJECT_SAMPLE_FACTORS - 01_020 Sample source:Norm. Area 01_20.raw (F63) | Injury severity:3X | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_20.raw SUBJECT_SAMPLE_FACTORS - 01_023 Sample source:Norm. Area 01_23.raw (F82) | Injury severity:3X | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_23.raw SUBJECT_SAMPLE_FACTORS - 01_028 Sample source:Norm. Area 01_28.raw (F55) | Injury severity:3X | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_28.raw SUBJECT_SAMPLE_FACTORS - 01_030 Sample source:Norm. Area 01_30.raw (F74) | Injury severity:3X | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_30.raw SUBJECT_SAMPLE_FACTORS - 01_032 Sample source:Norm. Area 01_32.raw (F73) | Injury severity:3X | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_32.raw SUBJECT_SAMPLE_FACTORS - 01_011 Sample source:Norm. Area 01_11.raw (F78) | Injury severity:3X | Sex:M | Batches:Batch 1 RAW_FILE_NAME=01_11.raw SUBJECT_SAMPLE_FACTORS - 01_012 Sample source:Norm. Area 01_12.raw (F54) | Injury severity:3X | Sex:M | Batches:Batch 1 RAW_FILE_NAME=01_12.raw SUBJECT_SAMPLE_FACTORS - 01_029 Sample source:Norm. Area 01_29.raw (F34) | Injury severity:3X | Sex:M | Batches:Batch 1 RAW_FILE_NAME=01_29.raw SUBJECT_SAMPLE_FACTORS - 01_014 Sample source:Norm. Area 01_14.raw (F38) | Injury severity:SHAM | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_14.raw SUBJECT_SAMPLE_FACTORS - 01_018 Sample source:Norm. Area 01_18.raw (F51) | Injury severity:SHAM | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_18.raw SUBJECT_SAMPLE_FACTORS - 01_019 Sample source:Norm. Area 01_19.raw (F53) | Injury severity:SHAM | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_19.raw SUBJECT_SAMPLE_FACTORS - 01_021 Sample source:Norm. Area 01_21.raw (F28) | Injury severity:SHAM | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_21.raw SUBJECT_SAMPLE_FACTORS - 01_031 Sample source:Norm. Area 01_31.raw (F56) | Injury severity:SHAM | Sex:F | Batches:Batch 1 RAW_FILE_NAME=01_31.raw SUBJECT_SAMPLE_FACTORS - 01_001 Sample source:Norm. Area 01_1.raw (F81) | Injury severity:SHAM | Sex:M | Batches:Batch 1 RAW_FILE_NAME=01_1.raw SUBJECT_SAMPLE_FACTORS - 01_002 Sample source:Norm. Area 01_2.raw (F44) | Injury severity:SHAM | Sex:M | Batches:Batch 1 RAW_FILE_NAME=01_2.raw SUBJECT_SAMPLE_FACTORS - 01_025 Sample source:Norm. Area 01_25.raw (F41) | Injury severity:SHAM | Sex:M | Batches:Batch 1 RAW_FILE_NAME=01_25.raw SUBJECT_SAMPLE_FACTORS - 01_026 Sample source:Norm. Area 01_26.raw (F80) | Injury severity:SHAM | Sex:M | Batches:Batch 1 RAW_FILE_NAME=01_26.raw SUBJECT_SAMPLE_FACTORS - 01_007 Sample source:Norm. Area 01_7.raw (F57) | Injury severity:SHAM | Sex:M | Batches:Batch 1 RAW_FILE_NAME=01_7.raw SUBJECT_SAMPLE_FACTORS - 02_010 Sample source:Norm. Area 02_10.raw (F43) | Injury severity:1X | Sex:F | Batches:Batch 2 RAW_FILE_NAME=02_10.raw SUBJECT_SAMPLE_FACTORS - 02_014 Sample source:Norm. Area 02_14.raw (F37) | Injury severity:1X | Sex:F | Batches:Batch 2 RAW_FILE_NAME=02_14.raw SUBJECT_SAMPLE_FACTORS - 02_026 Sample source:Norm. Area 02_26.raw (F45) | Injury severity:1X | Sex:F | Batches:Batch 2 RAW_FILE_NAME=02_26.raw SUBJECT_SAMPLE_FACTORS - 02_007 Sample source:Norm. Area 02_7.raw (F71) | Injury severity:1X | Sex:F | Batches:Batch 2 RAW_FILE_NAME=02_7.raw SUBJECT_SAMPLE_FACTORS - 02_011 Sample source:Norm. Area 02_11.raw (F27) | Injury severity:1X | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_11.raw SUBJECT_SAMPLE_FACTORS - 02_020 Sample source:Norm. Area 02_20.raw (F35) | Injury severity:1X | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_20.raw SUBJECT_SAMPLE_FACTORS - 02_021 Sample source:Norm. Area 02_21.raw (F67) | Injury severity:1X | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_21.raw SUBJECT_SAMPLE_FACTORS - 02_004 Sample source:Norm. Area 02_4.raw (F42) | Injury severity:1X | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_4.raw SUBJECT_SAMPLE_FACTORS - 02_008 Sample source:Norm. Area 02_8.raw (F52) | Injury severity:1X | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_8.raw SUBJECT_SAMPLE_FACTORS - 02_027 Sample source:Norm. Area 02_27.raw (F64) | Injury severity:3X | Sex:F | Batches:Batch 2 RAW_FILE_NAME=02_27.raw SUBJECT_SAMPLE_FACTORS - 02_005 Sample source:Norm. Area 02_5.raw (F58) | Injury severity:3X | Sex:F | Batches:Batch 2 RAW_FILE_NAME=02_5.raw SUBJECT_SAMPLE_FACTORS - 02_015 Sample source:Norm. Area 02_15.raw (F33) | Injury severity:3X | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_15.raw SUBJECT_SAMPLE_FACTORS - 02_016 Sample source:Norm. Area 02_16.raw (F47) | Injury severity:3X | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_16.raw SUBJECT_SAMPLE_FACTORS - 02_017 Sample source:Norm. Area 02_17.raw (F68) | Injury severity:3X | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_17.raw SUBJECT_SAMPLE_FACTORS - 02_018 Sample source:Norm. Area 02_18.raw (F26) | Injury severity:3X | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_18.raw SUBJECT_SAMPLE_FACTORS - 02_022 Sample source:Norm. Area 02_22.raw (F65) | Injury severity:3X | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_22.raw SUBJECT_SAMPLE_FACTORS - 02_023 Sample source:Norm. Area 02_23.raw (F66) | Injury severity:3X | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_23.raw SUBJECT_SAMPLE_FACTORS - 02_029 Sample source:Norm. Area 02_29.raw (F25) | Injury severity:3X | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_29.raw SUBJECT_SAMPLE_FACTORS - 02_031 Sample source:Norm. Area 02_31.raw (F75) | Injury severity:3X | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_31.raw SUBJECT_SAMPLE_FACTORS - 02_009 Sample source:Norm. Area 02_9.raw (F40) | Injury severity:3X | Sex:F | Batches:Batch 2 RAW_FILE_NAME=02_9.raw SUBJECT_SAMPLE_FACTORS - 02_013 Sample source:Norm. Area 02_13.raw (F30) | Injury severity:SHAM | Sex:F | Batches:Batch 2 RAW_FILE_NAME=02_13.raw SUBJECT_SAMPLE_FACTORS - 02_025 Sample source:Norm. Area 02_25.raw (F69) | Injury severity:SHAM | Sex:F | Batches:Batch 2 RAW_FILE_NAME=02_25.raw SUBJECT_SAMPLE_FACTORS - 02_028 Sample source:Norm. Area 02_28.raw (F70) | Injury severity:SHAM | Sex:F | Batches:Batch 2 RAW_FILE_NAME=02_28.raw SUBJECT_SAMPLE_FACTORS - 02_003 Sample source:Norm. Area 02_3.raw (F62) | Injury severity:SHAM | Sex:F | Batches:Batch 2 RAW_FILE_NAME=02_3.raw SUBJECT_SAMPLE_FACTORS - 02_012 Sample source:Norm. Area 02_12.raw (F60) | Injury severity:SHAM | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_12.raw SUBJECT_SAMPLE_FACTORS - 02_019 Sample source:Norm. Area 02_19.raw (F31) | Injury severity:SHAM | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_19.raw SUBJECT_SAMPLE_FACTORS - 02_002 Sample source:Norm. Area 02_2.raw (F48) | Injury severity:SHAM | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_2.raw SUBJECT_SAMPLE_FACTORS - 02_006 Sample source:Norm. Area 02_6.raw (F39) | Injury severity:SHAM | Sex:M | Batches:Batch 2 RAW_FILE_NAME=02_6.raw #COLLECTION CO:COLLECTION_SUMMARY Brain samples were harvested and collected following transcardial perfusion with CO:COLLECTION_SUMMARY phosphate buffer (0.1 M, pH 7.4) 24 hr post-TBI. The perfused whole brains were CO:COLLECTION_SUMMARY rapidly removed, and flash frozen in an isopentane-methanol ice slurry. Pieces CO:COLLECTION_SUMMARY of parietal cortices (5 mm x 2 mm) were dissected from partially thawed brains CO:COLLECTION_SUMMARY by removing the subcortical structures including the majority of white matter CO:COLLECTION_SUMMARY and stored at -80° C in microcentrifuge tubes. The cortices were then CO:COLLECTION_SUMMARY transferred to liquid nitrogen and manually pulverized with a pestle and mortar CO:COLLECTION_SUMMARY submerged in liquid nitrogen and aliquoted in ~10-30 mg tissue samples. CO:SAMPLE_TYPE Brain #TREATMENT TR:TREATMENT_SUMMARY Experimental batch 1 contained female (n = 16) and male (n = 10) to either sham TR:TREATMENT_SUMMARY procedure (n = 10), 1X, single mild traumatic brain injury (mTBI) (n = 8), and TR:TREATMENT_SUMMARY 3X, repetitive mTBI (n = 8) groups. Experimental batch 2 contained female (n = TR:TREATMENT_SUMMARY 11) and male (n = 17) to either sham procedure (n = 8), smTBI(n = 9), and rmTBI TR:TREATMENT_SUMMARY (n = 11) groups. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Experimental batch 1 and 2 aliquoted tissue samples were thawed simultane-ously SP:SAMPLEPREP_SUMMARY on ice prior to addition of solvent (IPA and Splash II Lipidomix in (1:3 v/v)) SP:SAMPLEPREP_SUMMARY to sep-arate lipids and small non-polar metabolites. LC-MS grade water was used SP:SAMPLEPREP_SUMMARY to prepare sample blanks, and pooled quality control (QC) samples were prepared SP:SAMPLEPREP_SUMMARY from 5 µL ali-quoted supernatant of all samples in the study. The brain and SP:SAMPLEPREP_SUMMARY solvent (1:4 w/v), and beads were placed in a Tissuelyser II for 8 min and SP:SAMPLEPREP_SUMMARY centrifuged at 16000 g for 7 min. The supernatant was collected for LC-MS. SP:SAMPLEPREP_SUMMARY Pooled quality control samples were formed from combining 6 µL aliquots of all SP:SAMPLEPREP_SUMMARY brain sample extracts. Sample blanks were prepared with the same procedure SP:SAMPLEPREP_SUMMARY except instead of a brain sample, 50 µL of LC-MS grade water was used. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY MS CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Thermo Accucore C30 (50 x 2.1mm,2.1um) CH:SOLVENT_A 40% water/60% acetonitrile; 10 mM ammonium formate; 0.1% formic acid CH:SOLVENT_B 10% acetonitrile/90% isopropanol; with 10 mM ammonium formate; 0.1% formic acid CH:FLOW_GRADIENT 0 minutes 80% A; 1 minute 40% A; 5 minutes 30% A; 5.5 minutes 15% A; 8 minutes CH:FLOW_GRADIENT 10% A; held 8.2 minutes to 10.5 minutes 0% A; 10.7 minutes 80% A; and held until CH:FLOW_GRADIENT 12 minutes CH:FLOW_RATE 0.40 mL/min CH:COLUMN_TEMPERATURE 50 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo ID-X Orbitrap Tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS LCMS Pos MS:MS_RESULTS_FILE ST003093_AN005062_Results.txt UNITS:m/z Has m/z:Yes Has RT:No RT units:No RT data #END