Abstract
Purpose :
To compare the developmental growth cone (GC) membrane with two models (Wnt3a and Zymosan) of induced optic nerve (ON) regeneration. To elucidate the lipid species that mark the non-regeneration to regeneration transition in the optic nerve.
Methods :
We performed high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the lipidome of GC from C57BL/6 mice across five age groups (E18, P0, P3, P6, and P9), n=6 with equal gender distribution. The lipids were extracted with the Bligh and Dyer method. For the Wnt3a and Zymosan models, optic nerves of 2-month-old mice were crushed ~1 mm behind the globe and subsequently injected with either saline, Wnt3a, or Zymosan intravitreally. Lipids were extracted from mouse ON at 3, 7, or 15 days post injections and analyzed in the same manner as growth cones. Data were normalized followed by ComBat batch correction. Classical receiving operator characteristic (ROC) curves were employed to identify lipid species with the greatest ability to detect regeneration.
Results :
Untargeted lipidomics on the same GC samples quantified 660 lipid species across 38 lipid classes in the GC membrane. We combined Wnt3a and Zymosan lipidomic datasets to find common lipid species and trends that differentiate regenerating optic nerves from control. We found differences in Ceramide of varying chain lengths, with the 18 and 24 acyl chain length showing higher in the growth permissive states. For the analysis of global lipid trends (biophysical and chemical properties), we ran lipid ontology analysis comparing regeneration and control groups. In comparing common significant lipid ontologies in regeneration in adult ON with early development (E18,P0) in growth cone, we discovered significant elevations in the number of annotations per term in “triacylglycerol”, “lipid storage”, “lipid droplet”, “glycerolipid”, and “headgroups with neutral charge” terms in both models.
Conclusions :
These findings provide evidence for the existence of a set of common lipid pathways and species that likely characterize the “growth non-permissive” to “growth permissive” transition in adult axons and in growth cone membranes in early development.
This is a 2020 ARVO Annual Meeting abstract.