title: Comprehensive Corticospinal Labeling with mu-crystallin Transgene Reveals Axon Regeneration after Spinal Cord Trauma in ngr1-/- Mice.
abstract: Spinal cord injury interrupts descending motor tracts and creates persistent functional deficits due to the absence of spontaneous axon regeneration. Of descending pathways, the corticospinal tract (CST) is thought to be the most critical for voluntary function in primates. Even with multiple tracer injections and genetic tools, the CST is visualized to only a minor degree in experimental studies. Here, we identify and validate the mu-crystallin (crym) gene as a high-fidelity marker of the CST. In transgenic mice expressing green fluorescent protein (GFP) under crym regulatory elements (crym-GFP), comprehensive and near complete CST labeling is achieved throughout the spinal cord. Bilateral pyramidotomy eliminated the 17,000 GFP-positive CST axons that were reproducibly labeled in brainstem from the spinal cord. We show that CST tracing with crym-GFP is 10-fold more efficient than tracing with biotinylated dextran amine (BDA). Using crym-GFP, we reevaluated the CST in mice lacking nogo receptor 1 (NgR1), a protein implicated in limiting neural repair. The number and trajectory of CST axons in ngr1(-/-) mice without injury was indistinguishable from ngr1(+/+) mice. After dorsal hemisection in the midthoracic cord, CST axons did not significantly regenerate in ngr1(+/+) mice, but an average of 162 of the 6000 labeled thoracic CST axons (2.68%) regenerated >100 μm past the lesion site in crym-GFP ngr1(-/-) mice. Although traditional BDA tracing cannot reliably visualize regenerating ngr1(-/-) CST axons, their regenerative course is clear with crym-GFP. Therefore the crym-GFP transgenic mouse is a useful tool for studies of CST anatomy in experimental studies of motor pathways.
  
  
  CONCLUSIONS
  Axon regeneration fails in the adult CNS, resulting in permanent functional deficits. Traditionally, inefficient extrinsic tracers such a biotinylated dextran amine (BDA) are used to label regenerating fibers after therapeutic intervention. We introduce crym-green fluorescent protein (GFP) transgenic mice as a comprehensive and specific tool with which to study the primary descending motor tract, the corticospinal tract (CST). CST labeling with crym-GFP is 10 times more efficient compared with BDA. The enhanced sensitivity afforded by crym-GFP revealed significant CST regeneration in NgR1 knock-out mice. Therefore, crym-GFP can be used as a standardized tool for future CST spinal cord injury studies.
authors.affiliation: Department of Neurology and Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06520.
authors.forename: Kathren L
authors.identifier: http://orcid.org/0000-0002-2956-4454
authors.initials: KL
authors.lastname: Fink
authors.affiliation: Department of Neurology and Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06520 william.cafferty@yale.edu stephen.strittmatter@yale.edu.
authors.forename: Stephen M
authors.identifier: http://orcid.org/0000-0001-8188-3092
authors.initials: SM
authors.lastname: Strittmatter
authors.affiliation: Department of Neurology and william.cafferty@yale.edu stephen.strittmatter@yale.edu.
authors.forename: William B J
authors.identifier: http://orcid.org/0000-0001-6263-2167
authors.initials: WB
authors.lastname: Cafferty
mesh_terms: D000578:Amidines / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D001369:Axons / Q000473:pathology; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D003459:Crystallins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D003911:Dextrans / Q000378:metabolism; D004195:Disease Models, Animal; D007839:Functional Laterality; D058851:GPI-Linked Proteins / Q000172:deficiency / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009185:Myelin Proteins / Q000172:deficiency* / Q000235:genetics; D009416:Nerve Regeneration / Q000235:genetics*; D000070816:Nogo Receptor 1; D011712:Pyramidal Tracts / Q000378:metabolism / Q000473:pathology*; D011956:Receptors, Cell Surface / Q000172:deficiency* / Q000235:genetics; D020127:Recovery of Function / Q000235:genetics; D013119:Spinal Cord Injuries / Q000150:complications* / Q000473:pathology; D000090803:mu-Crystallins
chemical_list: D000578:Amidines; C000716470:Crym protein, mouse; D003459:Crystallins; D003911:Dextrans; D058851:GPI-Linked Proteins; D005904:Glial Fibrillary Acidic Protein; D008164:Luminescent Proteins; D009185:Myelin Proteins; D000070816:Nogo Receptor 1; D011956:Receptors, Cell Surface; C419790:Rtn4r protein, mouse; C076397:biotinylated dextran amine; C031455:diamidino compound 253-50; D001710:Biotin; D000090803:mu-Crystallins
keywords: corticospinal tract; nogo receptor; regeneration; spinal cord injury; transgene