Summary of Study ST002524

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 PR001626. The data can be accessed directly via it's Project DOI: 10.21228/M8HT4B This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002524
Study Title	In situ microwave fixation provides an instantaneous snapshot of the brain metabolome - Part 1
Study Summary	We demonstrate exhaustion of glycogen and glucose and an increase in lactate production during conventional rapid brain resection prior to preservation by liquid nitrogen that is not observed with microwave fixation. Next, microwave fixation was employed to define the impact of brain glucose metabolism in the mouse model of streptozotocin-induced type 1 diabetes. Using both total pool and isotope tracing analyses, we identified global glucose hypometabolism in multiple regions of the mouse brain, evidenced by reduced 13C enrichment into glycogen, glycolysis, and the TCA cycle. Reduced glucose metabolism correlated with a marked decrease in GLUT2 expression and several metabolic enzymes in unique brain regions. In conclusion, our study supports the incorporation of microwave fixation to study terminal brain metabolism in rodent models.
Institute	University of Florida
Last Name	Sun
First Name	Ramon
Address	1200 Newell Drive, ARB
Email	ramonsun@ufl.edu
Phone	3522948407
Submit Date	2023-03-22
Raw Data Available	Yes
Raw Data File Type(s)	cdf
Analysis Type Detail	GC-MS
Release Date	2023-04-17
Release Version	1

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Project:

Project ID:	PR001626
Project DOI:	doi: 10.21228/M8HT4B
Project Title:	In situ microwave fixation provides an instantaneous snapshot of the brain metabolome
Project Summary:	The brain metabolome directly connects to brain physiology and neuronal function. Brain glucose metabolism is highly heterogeneous among brain regions and continues postmortem. Therefore, challenges remain to capture an accurate snapshot of the physiological brain metabolome in healthy and diseased rodent models. To overcome this barrier, we employed high-power focused microwave for the simultaneous euthanasia and fixation of mouse brain tissue to preserve metabolite pools prior to surgical removal and dissection of brain regions. We demonstrate exhaustion of glycogen and glucose and an increase in lactate production during conventional rapid brain resection prior to preservation by liquid nitrogen that is not observed with microwave fixation. Next, microwave fixation was employed to define the impact of brain glucose metabolism in the mouse model of streptozotocin-induced type 1 diabetes. Using both total pool and isotope tracing analyses, we identified global glucose hypometabolism in multiple regions of the mouse brain, evidenced by reduced 13C enrichment into glycogen, glycolysis, and the TCA cycle. Reduced glucose metabolism correlated with a marked decrease in GLUT2 expression and several metabolic enzymes in unique brain regions. In conclusion, our study supports the incorporation of microwave fixation to study terminal brain metabolism in rodent models.
Institute:	University of Florida
Department:	Biochemistry and Molecular Biology
Laboratory:	Sun lab
Last Name:	Sun
First Name:	Ramon
Address:	1200 Newell Drive, ARB
Email:	ramonsun@ufl.edu
Phone:	3522948407

Subject:

Subject ID:	SU002624
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	Tissue Region	preservation method
SA254517	CTX CP 2	cortex	cryopreservation
SA254518	CTX CP 1	cortex	cryopreservation
SA254519	CTX CP 3	cortex	cryopreservation
SA254520	CTX HPFM 2	cortex	focused microwave
SA254521	CTX HPFM 1	cortex	focused microwave
SA254522	CTX HPFM 3	cortex	focused microwave
SA254523	DVC CP 2	dorsal vagal complex	cryopreservation
SA254524	DVC CP 3	dorsal vagal complex	cryopreservation
SA254525	DVC CP 1	dorsal vagal complex	cryopreservation
SA254526	DVC HPFM 1	dorsal vagal complex	focused microwave
SA254527	DVC HPFM 2	dorsal vagal complex	focused microwave
SA254528	DVC HPFM 3	dorsal vagal complex	focused microwave
SA254511	Hipp CP 1	Hippocampus	cryopreservation
SA254512	Hipp CP 3	Hippocampus	cryopreservation
SA254513	Hipp CP 2	Hippocampus	cryopreservation
SA254514	Hipp HPFM 2	Hippocampus	focused microwave
SA254515	Hipp HPFM 3	Hippocampus	focused microwave
SA254516	Hipp HPFM 1	Hippocampus	focused microwave

Showing results 1 to 18 of 18

Showing results 1 to 18 of 18

Collection:

Collection ID:	CO002617
Collection Summary:	Mice were euthanized by microwave fixation system at 5kW for 0.6 seconds (MMW-05, Muromachi Kikai Company, Japan). Brain regions (HIPP, CTX, DVC), as well as muscle and liver, were dissected postmortem.
Sample Type:	Brain

Treatment:

Treatment ID:	TR002636
Treatment Summary:	We designed a two-arm study, where euthanasia occurs through either 1) direct decapitation or 2) focused microwave (Fig. 1). In both arms, we performed rapid dissection of the brain followed by cryopreservation in liquid nitrogen. The major difference between the two arms is the tissue fixation or enzyme inactivation step occurs either during cryopreservation for decapitation (CP, ~90 seconds) or focused microwave (FM, ~0.6 second).

Treatment ID:

TR002636

Treatment Summary:

We designed a two-arm study, where euthanasia occurs through either 1) direct decapitation or 2) focused microwave (Fig. 1). In both arms, we performed rapid dissection of the brain followed by cryopreservation in liquid nitrogen. The major difference between the two arms is the tissue fixation or enzyme inactivation step occurs either during cryopreservation for decapitation (CP, ~90 seconds) or focused microwave (FM, ~0.6 second).

Sample Preparation:

Sampleprep ID:	SP002630
Sampleprep Summary:	Brains were removed immediately post-mortem, and washed once with PBS, twice with diH2O, blotted dry, and snap frozen in liquid nitrogen. Other set of brains were snap frozen after microwave fixation as described above. The frozen tissues were pulverized to 10 μm particles in liquid N2 using a Freezer/Mill Cryogenic Grinder (SPEX SamplePrep). Brain regions were extracted with 1ml of 50% methanol in the grinder, while for muscle and liver twenty milligrams of each pulverized tissue were extracted in 1ml of 50% methanol and separated into polar (aqueous layer), and protein/DNA/RNA/glycogen pellet. The polar fraction was dried at 10-3 mBar using a SpeedVac (Thermo) followed by derivatization.

Sampleprep ID:

SP002630

Sampleprep Summary:

Brains were removed immediately post-mortem, and washed once with PBS, twice with diH2O, blotted dry, and snap frozen in liquid nitrogen. Other set of brains were snap frozen after microwave fixation as described above. The frozen tissues were pulverized to 10 μm particles in liquid N2 using a Freezer/Mill Cryogenic Grinder (SPEX SamplePrep). Brain regions were extracted with 1ml of 50% methanol in the grinder, while for muscle and liver twenty milligrams of each pulverized tissue were extracted in 1ml of 50% methanol and separated into polar (aqueous layer), and protein/DNA/RNA/glycogen pellet. The polar fraction was dried at 10-3 mBar using a SpeedVac (Thermo) followed by derivatization.

Combined analysis:

Analysis ID	AN004158
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 7890A
Column	Agilent HP5-MS (30m x 0.25mm, 0.25 um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5975C
Ion Mode	POSITIVE
Units	Relative abundance

Chromatography:

Chromatography ID:	CH003077
Instrument Name:	Agilent 7890A
Column Name:	Agilent HP5-MS (30m x 0.25mm, 0.25 um)
Column Temperature:	60-325
Flow Gradient:	na
Flow Rate:	0.69 ml/min
Solvent A:	na
Solvent B:	na
Chromatography Type:	GC

MS:

MS ID:	MS003905
Analysis ID:	AN004158
Instrument Name:	Agilent 5975C
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	GCMS protocols were similar to those described previously with a modified temperature gradient was used for GC: Initial temperature was 130degC, held for 4 minutes, rising at 6degC/minutes to 243degC, rising at 60degC/minutes to 280degC, held for 2 minutes. The electron ionization (EI) energy was set to 70 eV. Scan (m/z:50-800) and full scan mode were used for metabolomics analysis. Mass spectra were translated to relative metabolite abundance using the Automated Mass Spectral Deconvolution and Identification System (AMDIS) software matched to the FiehnLib metabolomics library (available through Agilent) for retention time and fragmentation pattern matching with a confidence score of > 80 (Fiehn, 2016; Fiehn et al., 2000; Kind et al., 2009). Data was further analyzed using the Data Extraction for Stable Isotope-labelled Metabolites (DEXSI) software package. Untargeted metabolomics data was normalized to total ion chromatogram. For glucose tracer raw data was exported and correction for natural abundance was done by IsoCorrectoR. Fractional enrichment of each metabolite was calculated as the relative abundance of each isotopologue relative to the sum of all other isotopologues. Mean enrichment was calculated as sum of fractional enrichment of labeled isotopologues (M1, M2, M3…). For principal component analysis, pathway impact analysis the online tool Metaboanlyst was used (https://www.metaboanalyst.ca/). Data was auto scaled and log transformed.
Ion Mode:	POSITIVE