Abstract
Purpose :
Ocular injuries of the anuran, Xenopus laevis, are uniquely befitted for studying the composition of retinal ganglion cells (RGC) due to functional recovery of axons disparate to human analogues. We profile the lipidome of optic nerves that were untreated or had monocular surgery of either an optic crush or sham surgery. Matching uninjured controls were also collected.
Methods :
Experiments were carried out in accordance to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research guidelines using 1 year old female transgenic frogs Tg(Islet2b: GFP) (n=144). Temporal post-injury phases of regenerative RGC axon regrowth was visualized confocally. Axons were anterogradely labeled with rhodamine dextran, intravitreally injected, and electroporated into the retina. Injured nerves were identified by punctate GFP expression of distal degenerating RGCs. Samples were extracted via Bligh and Dyer method and separated with a Vanquish liquid chromatography-tandem mass spectrometry. Untargeted analysis was done with 13 deuterated internal standards for class normalization and aligned using LipidSearch 5.0. Statistical analysis was done on Metaboanalyst 5.0.
Results :
Of 3134 lipids identified, variable importance scores >2 (partial least square discriminant analysis) and p < 0.05 were significant species between the crush and control nerves from 7, 12, 18, and 27 days post-injury (dpi); of which 25 were identified, of cholesterol ester, hexosyl ceramide, triglyceride, and monoglyceride sub-classes. Hierarchal cluster analysis of relative abundance of control and crush (Euclidean and Ward. D clustering algorithm) shows an incremental increase in the sphingomyelin sub-class of the crush groups up to 27 dpi. In contrast, controls at 27 dpi show a significant increase in hexosyl ceramide and ceramide phosphoethanolamine abundance from earlier diminished levels and with respect to the crush match.
Conclusions :
Axonal regrowth in Xenopus follow a regenerative timeline to exit the injury site at 7 dpi, cross the chiasm at 12 dpi, reach the tectal cup at 18 dpi, and institute synaptic connections in the tectal layers by 27 dpi. Stacked confocal images display regrowth at the optic chiasm at 12dpi. Preliminary analysis displaying temporal increases in sphingomyelin species in the ON of frogs after optic nerve crush confers myelin sheath formation over the tectum.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.