Summary of Study ST002776
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 PR001575. The data can be accessed directly via it's Project DOI: 10.21228/M83X51 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 | ST002776 |
Study Title | Zebrafish Optic Nerve Regeneration, Tectum Metabolomics - 3 Days Post Crush |
Study Summary | Optic nerve crush provides insight into the metabolome of the tectum. Regenerative model organisms provide key information regarding treatment degeneration in mammalian species; specifically, Zebrafish (Danio Rerio) have the capacity for successful adult optic nerve regeneration. Mammals, however, lack this intrinsic ability and undergo irreversible neurodegeneration seen in glaucoma, diabetes and other optic neuropathies. Optic nerve regeneration as well as the tectum metabolome are often studied to enhance regenerative research. Untargeted metabolomic studies within successful regenerative models are deficient. Evaluation of tectum tissue metabolomic changes in active zebrafish regeneration can elucidate prioritized metabolite pathways that can be targeted in mammalian systems for therapeutic development. Female and male (6 month to 1 year old) right Zebrafish (Tg(gap43:GFP)) optic nerves were crushed and the tecta were collected three days after. Contralateral, uninjured optic nerve tecta were collected as controls. The tissue was dissected from euthanized fish and frozen on dry ice. Samples were pooled for each category (female crush, female control, male crush, male control) and pooled at n = 10-12 to obtain sufficient metabolite concentrations for analysis. Optic nerve regeneration was verified by microscope visualization of GFP fluorescence. Metabolites were extracted using a Precellys Homogenizer and a serial extraction method: (1) 1:1 Methanol/Water and (2) 8:1:1 Acetonitrile/Methanol/Acetone. Metabolites were analyzed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. Metabolites were identified and quantified using Compound Discoverer 3.3 and isotopic internal metabolites standards. |
Institute | University of Miami |
Department | McKnight - Ophthalmology |
Laboratory | Bhattacharya Lab |
Last Name | Bhattacharya |
First Name | Sanjoy |
Address | 1638 NW 10th Avenue, Room 706-A, Miami, FL 33136 |
sbhattacharya@med.miami.edu | |
Phone | 3054824103 |
Submit Date | 2023-06-29 |
Num Groups | 2 |
Total Subjects | 67 |
Num Males | 36 |
Num Females | 31 |
Raw Data Available | Yes |
Raw Data File Type(s) | raw(Thermo) |
Analysis Type Detail | LC-MS |
Release Date | 2023-08-07 |
Release Version | 1 |
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Sample Preparation:
Sampleprep ID: | SP002889 |
Sampleprep Summary: | Tecta remained on dry ice to prevent metabolite degradation while the metabolite extraction was conducted. Tissues were transferred to 0.5mL Soft Tissue Lysing Kit Precellys tubes containing beads. Then, 84 µL of chilled 1:1 MeOH/H2O were added to Precellys tube. Pre-extraction internal standards were added to the tubes: 5µl of 1mg/ml Caffeine 13C6, 5µl of 1mg/ml D-Glucose 13C6, 5µl of 1mg/ml Oleic Acid 13C5, and 1µl of 5mg/mL Isoleucine 13C6 to each sample. Tissues were homogenized using Precellys 24 Touch. Cycle parameters: 2 cycles: 30 seconds homogenization at 4500 rpm, 10 seconds rest. Homogenate was transferred to a microcentrifuge tube and centrifuged at 18000xrcf for 20 min at 4°C. Then, collect supernatant and transfer pellet to Precellys Lysing Kit tube. Add 84uL of 8:1:1 Acetonitrile/Methanol/Acetone to pellet and add the rest of the pre-extraction internal standards: 5µl of 1mg/ml Caffeine 13C6, 5µl of 1mg/ml D-Glucose 13C6, 5µl of 1mg/ml Oleic Acid 13C5, 1µl of 5mg/mL Isoleucine 13C6. Final pre-extraction internal standards concentrations are 50μg/mL. Homogenization cycles were repeating using Precellys 24 Touch. Centrifuge as before and add second supernatant to first round of collected supernatant. Centrifuge at 1800xrcf for 20 min once more to remove any remaining tissue debris. Collect supernatant and dry supernatant in Speedvac. Two extraction blanks were prepared in the same manner as the biological samples. Dried samples were reconstituted immediately in 0.1% formic acid in 44.75µL of HPLC-MS grade water. Post-extraction internal standards were added: 25 µl of 5mg/ml Phenylalanine 13C6, 2.5 µl of .5mg/ml Uracil 13C 15N2, 1.25 µl of 1mg/ml Arginine 13C6, 1.25 µl of 1mg/ml Serine 13C3 to each sample. |