#METABOLOMICS WORKBENCH hormel101_20180717_163113_mwtab.txt DATATRACK_ID:1458 STUDY_ID:ST001021 ANALYSIS_ID:AN001675 PROJECT_ID:PR000683 VERSION 1 CREATED_ON July 19, 2018, 12:47 pm #PROJECT PR:PROJECT_TITLE Mayo Pilot and Feasibility: Characterization of metabolomics profile changes PR:PROJECT_TITLE during development of post-traumatic epilepsy PR:PROJECT_SUMMARY According to the report from the Centers for Disease Control and Prevention in PR:PROJECT_SUMMARY 2014, traumatic brain injury (TBI) accounts for 30% of all injury-related deaths PR:PROJECT_SUMMARY in the U.S. Developing epilepsy after severe head injury is as high as 40%-50% PR:PROJECT_SUMMARY in some settings. Importantly, many TBI victims develop epilepsy months or years PR:PROJECT_SUMMARY following the initial injury. However, it has not been fully identified how to PR:PROJECT_SUMMARY predict who will develop epilepsy and/or the underlying mechanism of post PR:PROJECT_SUMMARY traumatic epilesy (PTE) development. The main goal of this proposal is to PR:PROJECT_SUMMARY identify the metabolomics biomarker of TBI-induced epilepsy and to investigate PR:PROJECT_SUMMARY the underlying mechanism of the transition from TBI to PTE. To do so, we will PR:PROJECT_SUMMARY develop PTE using two TBI animal models: ferrous chloride injection model and PR:PROJECT_SUMMARY cortical undercut model. Once the brain damage is made, electroencephalogram PR:PROJECT_SUMMARY (EEG) and video monitoring will be performed to determine the onset point of PR:PROJECT_SUMMARY epilepsy. Then, metabolomics profile changes will be analyzed and compared PR:PROJECT_SUMMARY before and after PTE development. PR:INSTITUTE Mayo Clinic PR:LAST_NAME Su-youne PR:FIRST_NAME Chang PR:ADDRESS 200 1st Street SW Rochester, MN 55905, USA PR:EMAIL Chang.SuYoune@mayo.edu PR:PHONE 1-507-293-0511 #STUDY ST:STUDY_TITLE Characterization of metabolomics profile changes during development of ST:STUDY_TITLE post-traumatic epilepsy in Rat Cerebrospinal Fluid (part-II) ST:STUDY_SUMMARY Characterize the metabolomics profile changes during progression of the ST:STUDY_SUMMARY transition from traumatic brain injury (TBI) to post-traumatic epilepsy (PTE). ST:STUDY_SUMMARY To do so, three experiments will be performed. PTE animal model will be ST:STUDY_SUMMARY developed using ferrous chloride injections. Metabolomics profile changes will ST:STUDY_SUMMARY be obtained before TBI, after TBI, and after PTE development These temporal ST:STUDY_SUMMARY changes in metabolomics profile during the course of PTE development will be ST:STUDY_SUMMARY collected. We will also collect cerebrospinal fluid (CSF) at each time point. In ST:STUDY_SUMMARY addition, we will collect the brain tissue from the center of injury, around the ST:STUDY_SUMMARY injury, and from the non-injured area for mass spectrometry. In this study, Rat ST:STUDY_SUMMARY CSF is analyzed at end of study. ST:INSTITUTE Mayo Clinic ST:LAST_NAME Su-youne ST:FIRST_NAME Chang ST:ADDRESS 200 1st Street SW Rochester, MN 55905, USA ST:EMAIL Chang.SuYoune@mayo.edu ST:PHONE 1-507-293-0511 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 1 nC18-22aug17-001 groups:C chromatography=NC18 SUBJECT_SAMPLE_FACTORS 2 nC18-22aug17-002 groups:S chromatography=NC18 SUBJECT_SAMPLE_FACTORS 3 nC18-22aug17-003 groups:F chromatography=NC18 SUBJECT_SAMPLE_FACTORS 4 nC18-22aug17-004 groups:F chromatography=NC18 SUBJECT_SAMPLE_FACTORS 5 nC18-22aug17-005 groups:C chromatography=NC18 SUBJECT_SAMPLE_FACTORS 6 nC18-22aug17-006 groups:S chromatography=NC18 SUBJECT_SAMPLE_FACTORS 7 nC18-22aug17-007 groups:F chromatography=NC18 SUBJECT_SAMPLE_FACTORS 8 nC18-22aug17-008 groups:F chromatography=NC18 SUBJECT_SAMPLE_FACTORS 9 nC18-22aug17-009 groups:C chromatography=NC18 SUBJECT_SAMPLE_FACTORS 10 nC18-22aug17-010 groups:S chromatography=NC18 SUBJECT_SAMPLE_FACTORS 11 nC18-22aug17-011 groups:F chromatography=NC18 SUBJECT_SAMPLE_FACTORS 12 nC18-22aug17-012 groups:S chromatography=NC18 SUBJECT_SAMPLE_FACTORS 1 pC18-21aug17-001 groups:C chromatography=PC18 SUBJECT_SAMPLE_FACTORS 2 pC18-21aug17-002 groups:S chromatography=PC18 SUBJECT_SAMPLE_FACTORS 3 pC18-21aug17-003 groups:F chromatography=PC18 SUBJECT_SAMPLE_FACTORS 4 pC18-21aug17-004 groups:F chromatography=PC18 SUBJECT_SAMPLE_FACTORS 5 pC18-21aug17-005 groups:C chromatography=PC18 SUBJECT_SAMPLE_FACTORS 6 pC18-21aug17-006 groups:S chromatography=PC18 SUBJECT_SAMPLE_FACTORS 7 pC18-21aug17-007 groups:F chromatography=PC18 SUBJECT_SAMPLE_FACTORS 8 pC18-21aug17-008 groups:F chromatography=PC18 SUBJECT_SAMPLE_FACTORS 9 pC18-21aug17-009 groups:C chromatography=PC18 SUBJECT_SAMPLE_FACTORS 10 pC18-21aug17-010 groups:S chromatography=PC18 SUBJECT_SAMPLE_FACTORS 11 pC18-21aug17-011 groups:F chromatography=PC18 SUBJECT_SAMPLE_FACTORS 12 pC18-21aug17-012 groups:S chromatography=PC18 SUBJECT_SAMPLE_FACTORS 1 nhilic-18aug17-001_1 groups:C chromatography=nhilic SUBJECT_SAMPLE_FACTORS 2 nhilic-18aug17-002_1 groups:S chromatography=nhilic SUBJECT_SAMPLE_FACTORS 3 nhilic-18aug17-003_1 groups:F chromatography=nhilic SUBJECT_SAMPLE_FACTORS 4 nhilic-18aug17-004_1 groups:F chromatography=nhilic SUBJECT_SAMPLE_FACTORS 5 nhilic-18aug17-005_1 groups:C chromatography=nhilic SUBJECT_SAMPLE_FACTORS 6 nhilic-18aug17-006_1 groups:S chromatography=nhilic SUBJECT_SAMPLE_FACTORS 7 nhilic-18aug17-007_1 groups:F chromatography=nhilic SUBJECT_SAMPLE_FACTORS 8 nhilic-18aug17-008_1 groups:F chromatography=nhilic SUBJECT_SAMPLE_FACTORS 9 nhilic-18aug17-009_1 groups:C chromatography=nhilic SUBJECT_SAMPLE_FACTORS 10 nhilic-18aug17-010_1 groups:S chromatography=nhilic SUBJECT_SAMPLE_FACTORS 11 nhilic-18aug17-011_1 groups:F chromatography=nhilic SUBJECT_SAMPLE_FACTORS 12 nhilic-18aug17-012_1 groups:S chromatography=nhilic SUBJECT_SAMPLE_FACTORS 1 philic-17aug17-001 groups:C chromatography=philic SUBJECT_SAMPLE_FACTORS 2 philic-17aug17-002 groups:S chromatography=philic SUBJECT_SAMPLE_FACTORS 3 philic-17aug17-003 groups:F chromatography=philic SUBJECT_SAMPLE_FACTORS 4 philic-17aug17-004 groups:F chromatography=philic SUBJECT_SAMPLE_FACTORS 5 philic-17aug17-005 groups:C chromatography=philic SUBJECT_SAMPLE_FACTORS 6 philic-17aug17-006 groups:S chromatography=philic SUBJECT_SAMPLE_FACTORS 7 philic-17aug17-007 groups:F chromatography=philic SUBJECT_SAMPLE_FACTORS 8 philic-17aug17-008 groups:F chromatography=philic SUBJECT_SAMPLE_FACTORS 9 philic-17aug17-009 groups:C chromatography=philic SUBJECT_SAMPLE_FACTORS 10 philic-17aug17-010 groups:S chromatography=philic SUBJECT_SAMPLE_FACTORS 11 philic-17aug17-011 groups:F chromatography=philic SUBJECT_SAMPLE_FACTORS 12 philic-17aug17-012 groups:S chromatography=philic #COLLECTION CO:COLLECTION_SUMMARY "Rats will either be controls, injected with saline, or injected with ferrous CO:COLLECTION_SUMMARY chloride to influence PTE. Study Groupings: C=control, S=Saline treated, CO:COLLECTION_SUMMARY F=ferrous chloride treated Experimental Flow Day0: baseline pre TBI. Blood and CO:COLLECTION_SUMMARY CSF collected Day1: Surgery for TBI. Injections of Ferrous Cloride or Saline CO:COLLECTION_SUMMARY Day2: CSF collected Weeks1-3: montoring to determine PTE starting point. Blood CO:COLLECTION_SUMMARY and CSF collected 1 Month: montoring of PTE. Blood and CSF collected 2 Month: CO:COLLECTION_SUMMARY Animal is euthanized and blood, CSF, and tissue harvested CO:SAMPLE_TYPE CSF #TREATMENT TR:TREATMENT_SUMMARY "Rats will either be controls, injected with saline, or injected with ferrous TR:TREATMENT_SUMMARY chloride to influence PTE. Trauma-Induced Epilepsy Model: Ferrous chloride TR:TREATMENT_SUMMARY injection model: Ferrous chloride solution (5 μl of 100 mM with saline) will be TR:TREATMENT_SUMMARY injected at a rate of 0.5 μl/min through a Hamilton micro-syringe controlled by TR:TREATMENT_SUMMARY a micro-pump (UMP3, WPI, FL). Once the ferrous chloride solution injection is TR:TREATMENT_SUMMARY completed, the syringe will remain in position for 5 minutes, and then it will TR:TREATMENT_SUMMARY be removed slowly. The burr holes will be closed with light-curing dental TR:TREATMENT_SUMMARY cement. The dose of ferrous chloride injection was determined from prior TR:TREATMENT_SUMMARY published reports from mouse, rat, and cat. They all used 100 mM ferrous TR:TREATMENT_SUMMARY chloride aqueous solution and volumes were various: 1 μl for mouse,14 5 μl for TR:TREATMENT_SUMMARY rat (200-300 g),15 and 10 μl for cat (2-4 Kg).15 Video monitoring: The use of TR:TREATMENT_SUMMARY 24 x 7 video monitoring and review means that we do not have to rely on the rats TR:TREATMENT_SUMMARY having seizures during daily rounding or at some other time when a human happens TR:TREATMENT_SUMMARY to be present in the home cage. Normally, the video will be watched in time TR:TREATMENT_SUMMARY lapse, fast-forward mode to scan for potential seizures. The reviewer can then TR:TREATMENT_SUMMARY stop the video, rewind and watch the behavioral episode in real-time or slow TR:TREATMENT_SUMMARY motion to determine whether a seizure actually occurred. Behavioral seizures TR:TREATMENT_SUMMARY will be identified by any combination or sequence of the following: loss of TR:TREATMENT_SUMMARY postural control (opisthotonus), tonic flexion or extension of limbs or TR:TREATMENT_SUMMARY head/neck, and clonic movements of limbs or head/neck. Often, behavioral TR:TREATMENT_SUMMARY seizures in rats may be accompanied by drooling, urination and facial twitches, TR:TREATMENT_SUMMARY although these may not always be observable on video. In addition, seizures will TR:TREATMENT_SUMMARY likely be followed by a postictal phase, which may include a period of running, TR:TREATMENT_SUMMARY jumping and general agitation. Video monitoring cannot detect subclinical or TR:TREATMENT_SUMMARY electrographic seizures (i.e., seizures without a behavioral component). Video TR:TREATMENT_SUMMARY will be reviewed in this way for each rat in order to establish that a cortical TR:TREATMENT_SUMMARY injured rat does indeed have epilepsy, to establish the “typical seizure” TR:TREATMENT_SUMMARY pattern in each rat, and to help establish a seizure frequency baseline, TR:TREATMENT_SUMMARY although it is understood that video monitoring alone might occasionally miss a TR:TREATMENT_SUMMARY seizure. EEG monitoring: To prevent imaging distortion and ferromagnetic TR:TREATMENT_SUMMARY interference, graphite carbon electrodes will be fabricated and/or purchased. A TR:TREATMENT_SUMMARY total of five electrodes will be implanted for EEG monitoring on the skull. EEG TR:TREATMENT_SUMMARY will be monitored with the Open EPhys System.18 While EEG recording, EEG TR:TREATMENT_SUMMARY electrodes will be connected to wires attached to the ceiling of a cage. In TR:TREATMENT_SUMMARY trauma-induced epilepsy rats, spontaneous neural activity will be recorded using TR:TREATMENT_SUMMARY a wide bandwidth (0-9 kHz) recording system. Post-analysis will be used to TR:TREATMENT_SUMMARY identify epilepsy signals." #SAMPLEPREP SP:SAMPLEPREP_SUMMARY "large scale profiling of rat cerebral spinal fluid The brain tissue and CSF SP:SAMPLEPREP_SUMMARY will be collected for mass spectrometry. To prepare samples, proteins will be SP:SAMPLEPREP_SUMMARY removed from collected dialysates by adding cold methanol:water (8:1, v/v) SP:SAMPLEPREP_SUMMARY mixture containing 5.0 μg internal standard (IS), myristic-d27 acid, at ambient SP:SAMPLEPREP_SUMMARY temperature. Samples will be vortexed for 1 min, incubated on ice for 15 min, SP:SAMPLEPREP_SUMMARY and then centrifuged. The supernatant will be completely dried in a SpeedVac, SP:SAMPLEPREP_SUMMARY and the lyophilized sample will be subsequently methoxiaminated using 20 μl of SP:SAMPLEPREP_SUMMARY a 20 mg/ml solution of methoxyamine hydrochloride in pyridine at 30°C for 90 SP:SAMPLEPREP_SUMMARY min and derivatized using 80 μL of N-methyl-N-trimethylsilyltrifluoroacetamide SP:SAMPLEPREP_SUMMARY with 1% trimethylchloro-silane (MSTFA + 1% TMCS, Pierce) at 37°C for 30 min." #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters Acquity HSS C18 (150 x 2.1mm, 1.8um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_RESULTS_FILE ST001021_AN001675_Results.txt UNITS:intensity #END