Genetic Background Of Optic Nerve Regeneration In BXD Mouse Strains

PURPOSE:This study is designed to identify the influences of genetic background on retinal ganglion cell axon regeneration after optic nerve crush using BXD recombinant inbred(RI)mouse strains.METHODS:To study optic nerve regeneration,we constructed a short hairpin RNA(sh RNA)plasmid containing the phosphatase and tensin homologue(Pten)gene-targeting sequences using adenovirus 2(AAV2)as the vector.The plasmid was transfected into mouse retinal ganglion cells through intravitreal injection and the expression of Pten was silenced.Two weeks after the gene therapy,optic nerve was damaged by optic nerve crush(ONC).Intravitreal injection of Zymosan and 8-CPT-cyclic adenine mimetics(8-CPT-c AMP)was performed immediately after ONC to induce a mild intraocular inflammation.This combined treatment was applied to 9 strains of mice(C57BL/6J,DBA/2J,BXD11,BXD29,BXD31,BXD38,BXD40,BXD75,BXD102)to induce optic nerve regeneration.The regenerating axons were labeled by cholera toxin B and were examined 2 weeks after ONC.Quantititive examination of the axon regeneration include measureing the number of axons at 0.5 mm and 1 mm away from the crush site in the optic nerve,and the distances that the longest 5 regenerated axons and the longest single regenerating axon traveled along the nerve.The measurements were analysed in the Gene Network database.Quantitative trait locus(QTL)were mapped to determine the genomic loci involved in the modulation of optic nerve regeneration in the BXD strain set.Single nucleotide polymorphism(SNP)analysis,cis quantitative trait locus(cis-QTL)analysis,and protein function prediction analysis(SIFT)were applied to identify candidate genes within the QTL region.Beyond that,the above analysis was performed on known regeneration-related genes to identify candidate genes that may have impact on optic nerve regeneration in the BXD RI mouse strains.RESULTS:The analysis revealed a considerable amount of differential axonal regeneration across the seven BXD strains and the parental strains.Among the mice included in this study,BXD29 has the most robust regeneration and BXD102 has the least regeneration.There was a statistically significant difference(3~12-fold)in the number of regenerated axons(236.1±24.4 axons in BXD102 vs 759.8±79.2 axons in BXD29 at 0.5 mm from crush,P=0.014;1 ± 0 axons in BXD102 vs 12.6 ± 0.6 axons in BXD29 at 1 mm from crush,P=0.007).There were also statistically significant differences(2-2.5-fold)in the distance axons traveled across BXD strains(787.2±46.5 ?m in BXD102 vs 2025.5±223.3 ?m in BXD29,looking at the longest five axons,P=0.014;1107 ± 40.6 ?m in BXD102 vs 2386.8 ± 162.6 ?m in BXD29,looking at the longest axon,P=0.014).Axon regeneration of the parental strains(C57BL/6J and DBA/2J)was about the middle level of the BXD strains.Quantititive trait locus(QTL)analysis revealed a that chromosome 11,from 69 to 70 megabase,is involved in the modulation of the regenerated axons numbers.Further analysis of this region identified 17 candidate genes(Wrap53,Per1,Sat2,Alox8,Hes7,Kcnab3,Mir467 f,Ef4a1,Tmem102,Chd3 os,Alox12b,Tmem107,Aloxe3,Cyb5d1,Tmem95,Slc2a4,Senp3).In addition,possible roles of 3 known regeneration-related genes(Fgf2,Mapk10 and Rtn4)were identified in the regulation of optic nerve regeneration in BXD RI mouse strains.CONCLUSION:Optic nerve regeneration is a complex trait.Genetic background may have a profound effect on axonal regeneration in mouse optic nerves.A QTL of chromosome 11(69-70 megabase)is identified to be involved in genetic regulation of optic nerve regeneration in mice.Further investigation of specific roles and mechanism of the candidate genes are recommended.