Summary of Study ST002444

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, 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.


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.

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Study IDST002444
Study TitleZebrafish Optic Nerve Regeneration Metabolomics - 3 Days Post Crush
Study SummaryZebrafish (Danio Rerio) have the capacity for successful adult optic nerve regeneration. In contrast, mammals lack this intrinsic ability and undergo irreversible neurodegeneration seen in glaucoma and other optic neuropathies. Optic nerve regeneration is often studied using optic nerve crush, a mechanical neurodegenerative model. Untargeted metabolomic studies within successful regenerative models are deficient. Evaluation of tissue metabolomic changes in active zebrafish optic nerve 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 collected three days after. Contralateral, uninjured optic nerves 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 = 31 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.
University of Miami
DepartmentMcKnight - Ophthalmology
LaboratoryBhattacharya Lab
Last NameBhattacharya
First NameSanjoy
Address1638 NW 10th Avenue, Room 706-A, Miami, FL 33136
Submit Date2022-12-19
Num Groups2
Total Subjects67
Num Males36
Num Females31
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2023-01-25
Release Version1
Sanjoy Bhattacharya Sanjoy Bhattacharya application/zip

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Sample Preparation:

Sampleprep ID:SP002539
Sampleprep Summary:Metabolite extraction from samples was carried out quickly while keeping optic nerve tissues on dry ice to prevent metabolite degradation. Tissues were transferred to 0.5mL Soft Tissue Lysing Kit Precellys tubes containing beads. Add 84 µL of chilled 1:1 MeOH/H2O to Precellys tube. Pre-extraction internal standards were added to the tubes. Add pre-extraction internal standards: 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. Transfer homogenate to microcentrifuge tube and centrifuge at 18000xrcf for 20 min at 4°C. 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.