Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA064195

Local Sample ID:	nC18-07sep17-009
Subject ID:	SU001061
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

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Subject:

Subject ID:	SU001061
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
nC18-07sep17-009	SA064195	FL010946	F	Groups
nC18-07sep17-009	SA064195	FL010946	Cortex	Tissues

Collection:

Collection ID:	CO001055
Collection Summary:	Rats will either be controls, injected with saline, or injected with ferrous chloride to influence PTE. Also one of the ferrous chloride treated rats was repeated (R_no_cx, R_no_hp, R_inj_cx, R_inj_hp). Study Groupings: C_no_cx = control, no injections, cortex tissue C_no_hp = control, no injections, hippocampus tissue S_no_cx = saline treated, cortex tissue not near injection site S_no_hp = saline treated, hippocampus tissue not near injection site S_inj_cx = saline treated, cortex tissue at injection site S_inj_hp = saline treated, hippocampus tissue at injection site F_no_cx = ferrous chloride treated, cortex tissue not near injection site F_no_hp = ferrou chloride treated, hippocampus tissue not near injection site F_inj_cx = ferrous chloride treated, cortex tissue at injection site F_inj_hp = ferrous chloride treated, hippocampus tissue at injection site Experimental Flow Day0: baseline pre TBI. Blood and CSF collected Day1: Surgery for TBI. Injections of Ferrous Cloride or Saline Day2: CSF collected Weeks1-3: montoring to determine PTE starting point. Blood and CSF collected 1 Month: montoring of PTE. Blood and CSF collected 2 Month: Animal is euthanized and blood, CSF, and tissue harvested
Sample Type:	Brain

Treatment:

Treatment ID:	TR001075
Treatment Summary:	Rats will either be controls, injected with saline, or injected with ferrous chloride to influence PTE. Trauma-Induced Epilepsy Model: Ferrous chloride injection model: Ferrous chloride solution (5 μl of 100 mM with saline) will be injected at a rate of 0.5 μl/min through a Hamilton micro-syringe controlled by a micro-pump (UMP3, WPI, FL). Once the ferrous chloride solution injection is completed, the syringe will remain in position for 5 minutes, and then it will be removed slowly. The burr holes will be closed with light-curing dental cement. The dose of ferrous chloride injection was determined from prior published reports from mouse, rat, and cat. They all used 100 mM ferrous chloride aqueous solution and volumes were various: 1 μl for mouse,14 5 μl for rat (200-300 g),15 and 10 μl for cat (2-4 Kg).15 Video monitoring: The use of 24 x 7 video monitoring and review means that we do not have to rely on the rats having seizures during daily rounding or at some other time when a human happens to be present in the home cage. Normally, the video will be watched in time lapse, fast-forward mode to scan for potential seizures. The reviewer can then stop the video, rewind and watch the behavioral episode in real-time or slow motion to determine whether a seizure actually occurred. Behavioral seizures will be identified by any combination or sequence of the following: loss of postural control (opisthotonus), tonic flexion or extension of limbs or head/neck, and clonic movements of limbs or head/neck. Often, behavioral seizures in rats may be accompanied by drooling, urination and facial twitches, although these may not always be observable on video. In addition, seizures will likely be followed by a postictal phase, which may include a period of running, jumping and general agitation. Video monitoring cannot detect subclinical or electrographic seizures (i.e., seizures without a behavioral component). Video will be reviewed in this way for each rat in order to establish that a cortical injured rat does indeed have epilepsy, to establish the “typical seizure” pattern in each rat, and to help establish a seizure frequency baseline, although it is understood that video monitoring alone might occasionally miss a seizure. EEG monitoring: To prevent imaging distortion and ferromagnetic interference, graphite carbon electrodes will be fabricated and/or purchased. A total of five electrodes will be implanted for EEG monitoring on the skull. EEG will be monitored with the Open EPhys System.18 While EEG recording, EEG electrodes will be connected to wires attached to the ceiling of a cage. In trauma-induced epilepsy rats, spontaneous neural activity will be recorded using a wide bandwidth (0-9 kHz) recording system. Post-analysis will be used to identify epilepsy signals.

Treatment ID:

TR001075

Treatment Summary:

Rats will either be controls, injected with saline, or injected with ferrous chloride to influence PTE. Trauma-Induced Epilepsy Model: Ferrous chloride injection model: Ferrous chloride solution (5 μl of 100 mM with saline) will be injected at a rate of 0.5 μl/min through a Hamilton micro-syringe controlled by a micro-pump (UMP3, WPI, FL). Once the ferrous chloride solution injection is completed, the syringe will remain in position for 5 minutes, and then it will be removed slowly. The burr holes will be closed with light-curing dental cement. The dose of ferrous chloride injection was determined from prior published reports from mouse, rat, and cat. They all used 100 mM ferrous chloride aqueous solution and volumes were various: 1 μl for mouse,14 5 μl for rat (200-300 g),15 and 10 μl for cat (2-4 Kg).15 Video monitoring: The use of 24 x 7 video monitoring and review means that we do not have to rely on the rats having seizures during daily rounding or at some other time when a human happens to be present in the home cage. Normally, the video will be watched in time lapse, fast-forward mode to scan for potential seizures. The reviewer can then stop the video, rewind and watch the behavioral episode in real-time or slow motion to determine whether a seizure actually occurred. Behavioral seizures will be identified by any combination or sequence of the following: loss of postural control (opisthotonus), tonic flexion or extension of limbs or head/neck, and clonic movements of limbs or head/neck. Often, behavioral seizures in rats may be accompanied by drooling, urination and facial twitches, although these may not always be observable on video. In addition, seizures will likely be followed by a postictal phase, which may include a period of running, jumping and general agitation. Video monitoring cannot detect subclinical or electrographic seizures (i.e., seizures without a behavioral component). Video will be reviewed in this way for each rat in order to establish that a cortical injured rat does indeed have epilepsy, to establish the “typical seizure” pattern in each rat, and to help establish a seizure frequency baseline, although it is understood that video monitoring alone might occasionally miss a seizure. EEG monitoring: To prevent imaging distortion and ferromagnetic interference, graphite carbon electrodes will be fabricated and/or purchased. A total of five electrodes will be implanted for EEG monitoring on the skull. EEG will be monitored with the Open EPhys System.18 While EEG recording, EEG electrodes will be connected to wires attached to the ceiling of a cage. In trauma-induced epilepsy rats, spontaneous neural activity will be recorded using a wide bandwidth (0-9 kHz) recording system. Post-analysis will be used to identify epilepsy signals.

Sample Preparation:

Sampleprep ID:	SP001068
Sampleprep Summary:	large scale profiling of rat cortex and hippocampus brian tissue. The brain tissue and CSF will be collected for mass spectrometry. To prepare samples, proteins will be removed from collected dialysates by adding cold methanol:water (8:1, v/v) mixture containing 5.0 μg internal standard (IS), myristic-d27 acid, at ambient temperature. Samples will be vortexed for 1 min, incubated on ice for 15 min, and then centrifuged. The supernatant will be completely dried in a SpeedVac, and the lyophilized sample will be subsequently methoxiaminated using 20 μl of a 20 mg/ml solution of methoxyamine hydrochloride in pyridine at 30°C for 90 min and derivatized using 80 μL of N-methyl-N-trimethylsilyltrifluoroacetamide with 1% trimethylchloro-silane (MSTFA + 1% TMCS, Pierce) at 37°C for 30 min.

Sampleprep ID:

SP001068

Sampleprep Summary:

large scale profiling of rat cortex and hippocampus brian tissue. The brain tissue and CSF will be collected for mass spectrometry. To prepare samples, proteins will be removed from collected dialysates by adding cold methanol:water (8:1, v/v) mixture containing 5.0 μg internal standard (IS), myristic-d27 acid, at ambient temperature. Samples will be vortexed for 1 min, incubated on ice for 15 min, and then centrifuged. The supernatant will be completely dried in a SpeedVac, and the lyophilized sample will be subsequently methoxiaminated using 20 μl of a 20 mg/ml solution of methoxyamine hydrochloride in pyridine at 30°C for 90 min and derivatized using 80 μL of N-methyl-N-trimethylsilyltrifluoroacetamide with 1% trimethylchloro-silane (MSTFA + 1% TMCS, Pierce) at 37°C for 30 min.

Combined analysis:

Analysis ID	AN001676	AN001677	AN001678	AN001679
Analysis type	MS	MS	MS	MS
Chromatography type	HILIC	HILIC	Reversed phase	Reversed phase
Chromatography system	Agilent 1290 Infinity	Agilent 1290 Infinity	Agilent 1290 Infinity	Agilent 1290 Infinity
Column	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)	Waters Acquity HSS C18 (150 x 2.1mm,1.8um)	Waters Acquity HSS C18 (150 x 2.1mm,1.8um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	QTOF	QTOF	QTOF	QTOF
MS instrument name	Agilent 6550 QTOF	Agilent 6550 QTOF	Agilent 6550 QTOF	Agilent 6550 QTOF
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	intensity	intensity	intensity	intensity

Chromatography:

Chromatography ID:	CH001179
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Chromatography Type:	HILIC

Chromatography ID:	CH001180
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters Acquity HSS C18 (150 x 2.1mm,1.8um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001551
Analysis ID:	AN001676
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	POSITIVE

MS ID:	MS001552
Analysis ID:	AN001677
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	NEGATIVE

MS ID:	MS001553
Analysis ID:	AN001678
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	POSITIVE

MS ID:	MS001554
Analysis ID:	AN001679
Instrument Name:	Agilent 6550 QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	NEGATIVE