Robust optic nerve regeneration in a mouse mutant

Abstract

Purpose : Previously we examined the effects of genetic background on optic nerve regeneration (ONR) using the BXD inbreed strain set. One strain, BXD29-Tlr4^lps-2J/J, displayed robust axonal regeneration. This strain is known to have acquired a mutation in TRL4 since its initial production in 1976. The present study is designed to determine if the mutation in TRL4 is responsible for the profound regeneration.

Methods : The present study examined optic nerve regeneration in BXD29-Tlr4^lps-2J/J mice and BXD29/Ty (the cryopreserved original strain) mice, the F1 crosses and the F2 crosses. ONR protocols include knockdown of Pten and intravitreal injection of zymosan and cAMP. To identify the role of the known mutant gene Tlr4, we examined the effects of this treatment following optic nerve crush. Two days before sacrifice, Alexa Fluor® 647 Conjugated Cholera Toxin B was injected into the vitreous and at 14 days after crush, the optic nerves were fixed and cleared in FocusClear. The amount of regeneration was quantified by the number of axons at 0.5 mm and 1 mm from the crush, and the distance that the longest 5 axons and single axon had reached down the nerve. To explore other candidate genes, we explored the full genomic sequences of both BXD29-Tlr4^lps-2J/J and BXD29/Ty. All SNPs and INDELs were called and compared between the two strains to identify possible mutations that may have impact on the regeneration.

Results : There was a considerable amount of axonal growth on BXD29-Tlr4^lps-2J/J but not the BXD29/Ty. All the F1s and F2s have shown similarly robust regeneration as the BXD29-Tlr4^lps-2J/, indicating that the genetic element that is regulating the regeneration could be autosomal dominant. There is no significant difference in the amount of regeneration between the homozygote F2s (+/+ for wild type Tlr4) and the heterozygote F2s (+/- for mutant Tlr4), indicating that the mutation of Tlr4 is not responsible for this robust regeneration response. Since the two sub-strains have been separated for over 40 years, other mutations may have occurred. After whole genome analysis, we have identified more than 5,000 variants between the two sub-strains with 293 variants in the protein coding region. We are currently evaluating the segregation of these SNPs to identify the genomic elements responsible for the increased regeneration.

Conclusions : The strong optic nerve regeneration found in the BXD29-Tlr4^lps-2J/J mice was not caused by the mutation in Tlr4.

This is a 2021 ARVO Annual Meeting abstract.