PR001575 (Project)

Description:Zebrafish (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. Currently, 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 to 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. The associated retinas and tecta were also collected under the same conditions for metabolic analysis. Contralateral, uninjured optic nerves, retinas and tecta were collected as controls. The three tissue types (optic nerve, retina, and tectum) were dissected from euthanized fish and frozen on dry ice. Optic nerve 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. Retina and tectum samples were pooled using the same categories (female crush, female control, male crush, male control) at n = 10-12. 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 metabolite standards.