Summary of Study ST002525
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 | ST002525 |
| Study Title | In situ microwave fixation provides an instantaneous snapshot of the brain metabolome - Part 2 |
| 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 |
| 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 |
Select appropriate tab below to view additional metadata details:
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: | SU002625 |
| 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 | Treatment | Tissue Region |
|---|---|---|---|
| SA254537 | CTX T1DM 4 | STZ | cortex |
| SA254538 | CTX T1DM 3 | STZ | cortex |
| SA254539 | CTX T1DM 5 | STZ | cortex |
| SA254540 | CTX T1DM 7 | STZ | cortex |
| SA254541 | CTX T1DM 2 | STZ | cortex |
| SA254542 | CTX T1DM 6 | STZ | cortex |
| SA254543 | CTX T1DM 1 | STZ | cortex |
| SA254544 | DVC T1DM 4 | STZ | dorsal vagal complex |
| SA254545 | DVC T1DM 3 | STZ | dorsal vagal complex |
| SA254546 | DVC T1DM 5 | STZ | dorsal vagal complex |
| SA254547 | DVC T1DM 6 | STZ | dorsal vagal complex |
| SA254548 | DVC T1DM 8 | STZ | dorsal vagal complex |
| SA254549 | DVC T1DM 7 | STZ | dorsal vagal complex |
| SA254550 | DVC T1DM 1 | STZ | dorsal vagal complex |
| SA254551 | DVC T1DM 2 | STZ | dorsal vagal complex |
| SA254529 | Hipp T1DM 4 | STZ | Hippocampus |
| SA254530 | Hipp T1DM 2 | STZ | Hippocampus |
| SA254531 | Hipp T1DM 1 | STZ | Hippocampus |
| SA254532 | Hipp T1DM 5 | STZ | Hippocampus |
| SA254533 | Hipp T1DM 3 | STZ | Hippocampus |
| SA254534 | Hipp T1DM 6 | STZ | Hippocampus |
| SA254535 | Hipp T1DM 8 | STZ | Hippocampus |
| SA254536 | Hipp T1DM 7 | STZ | Hippocampus |
| SA254560 | CTX Vehicle 6 | vehicle | cortex |
| SA254561 | CTX Vehicle 7 | vehicle | cortex |
| SA254562 | CTX Vehicle 1 | vehicle | cortex |
| SA254563 | CTX Vehicle 5 | vehicle | cortex |
| SA254564 | CTX Vehicle 4 | vehicle | cortex |
| SA254565 | CTX Vehicle 2 | vehicle | cortex |
| SA254566 | CTX Vehicle 3 | vehicle | cortex |
| SA254567 | DVC Vehicle 1 | vehicle | dorsal vagal complex |
| SA254568 | DVC Vehicle 2 | vehicle | dorsal vagal complex |
| SA254569 | DVC Vehicle 5 | vehicle | dorsal vagal complex |
| SA254570 | DVC Vehicle 4 | vehicle | dorsal vagal complex |
| SA254571 | DVC Vehicle 6 | vehicle | dorsal vagal complex |
| SA254572 | DVC Vehicle 7 | vehicle | dorsal vagal complex |
| SA254573 | DVC Vehicle 8 | vehicle | dorsal vagal complex |
| SA254574 | DVC Vehicle 3 | vehicle | dorsal vagal complex |
| SA254552 | Hipp Vehicle 7 | vehicle | Hippocampus |
| SA254553 | Hipp Vehicle 8 | vehicle | Hippocampus |
| SA254554 | Hipp Vehicle 6 | vehicle | Hippocampus |
| SA254555 | Hipp Vehicle 1 | vehicle | Hippocampus |
| SA254556 | Hipp Vehicle 3 | vehicle | Hippocampus |
| SA254557 | Hipp Vehicle 4 | vehicle | Hippocampus |
| SA254558 | Hipp Vehicle 2 | vehicle | Hippocampus |
| SA254559 | Hipp Vehicle 5 | vehicle | Hippocampus |
| Showing results 1 to 46 of 46 |
Collection:
| Collection ID: | CO002618 |
| 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: | TR002637 |
| 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: | SP002631 |
| 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 | AN004159 |
|---|---|
| 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: | CH003078 |
| 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: | MS003906 |
| Analysis ID: | AN004159 |
| 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 |
