Abstract
Purpose :
Mechanical forces determine the movement of regenerating retinal ganglion cell (RGC) axons, facilitating their navigation to intended targets within the brain. Mechanosensitive (MS) ion channels detect mechanical forces within cell membranes and play a potential role in transducing these stimuli. We investigated the expression of 12 different MS channels (PKD2, PIEZO1, PIEZO2, ASIC2, alpha ENaC, TRPC2, TRPC3, TRPC5, TRPA1, TRPV4, TMC1, TRPM2) in crushed and axon regenerating optic nerves of the mouse to determine whether they are associated with regenerating axons.
Methods :
Two groups of C57BL/6J mice (n=6, 2 months old, even sex distribution) were studied: the first group underwent pharmacologically induced axon regeneration via AAV-mediated ciliary neurotrophic factor (CNTF) followed by optic nerve crush (ONC) 2 weeks later, and the second (control) group underwent ONC without treatment. Prior to euthanasia, both groups received fluorophore-conjugated CTB intravitreal injections. Optic nerves were extracted, prepared for immunohistochemistry (IHC) using polyclonal antibodies, and imaged using a confocal light microscope. Fluorescence data was analyzed using a densitometric scan and ImageJ software to calculate the mean and standard deviation of MS channel expression levels. The means of both groups were compared using a student’s t-test, and IHC data was compared with Gene Expression Omnibus (GEO) transcriptomic datasets.
Results :
Image analysis using ImageJ subjected to a t-test suggested significant differences (p ≤ 0.05) between crushed and axon regenerating optic nerves in certain channels. Of the 12 MS channels evaluated, 5 yielded similar expression levels between crushed and regenerating optic nerves. Of the other 7, TRPC3, TRPC5, TMC1, and PIEZO2 demonstrated upregulation in regenerated optic nerves, whereas TRPM2, ASIC1, and ASIC2 demonstrated downregulation. Transcriptomic dataset analysis was consistent with these results with a positive fold change in TRPC3, TRPC5, and TMC1 and a negative fold change in TRPM2 and ASIC1 in optic nerve tissues.
Conclusions :
Our findings suggest that MS channels are differentially present in crushed versus axon regenerating optic nerves. Future experiments will explore whether specific channel levels are associated with the looping or misdirection of axons, guiding strategies for directing regenerating axons to the correct brain regions.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.