title: Delayed grafting of BDNF and NT-3 producing fibroblasts into the injured spinal cord stimulates sprouting, partially rescues axotomized red nucleus neurons from loss and atrophy, and provides limited regeneration.
abstract: Ex vivo gene therapy, utilizing modified fibroblasts that deliver BDNF or NT-3 to the acutely injured spinal cord, has been shown to elicit regeneration and recovery of function in the adult rat. Delayed grafting into the injured spinal cord is of great clinical interest as a model for treatment of chronic injury but may pose additional obstacles that are not present after acute injury, such as the need to remove an established scar, increased retrograde cell loss and/or atrophy, and diminished capacity for regeneration by neurons which may be doubly injured. The purpose of the present study was to determine if delayed grafting of neurotrophin secreting fibroblasts would have anatomical effects similar to those seen in acute grafting models. We grafted a mixture of BDNF and NT-3 producing fibroblasts or control fibroblasts into a complete unilateral cervical hemisection after a 6-week delay. Fourteen weeks after delayed grafting we found that both the neurotrophin secreting fibroblasts and control fibroblasts survived, but that only the neurotrophin secreting grafts provided a permissive environment for host axon growth, as indicated by immunostaining for RT-97, a marker for axonal neurofilaments, GAP-43, a marker for elongating axons, CGRP, a marker for dorsal root axons, and 5-HT, a marker for raphe spinal axons, within the graft. Anterograde tracing of the uninjured vestibulospinal tract showed growth into neurotrophin producing transplants but not into control grafts, while anterograde tracing of the axotomized rubrospinal tract showed a small number of regenerating axons within the genetically modified grafts, but none in control grafts. The neurotrophin expressing grafts, but not the control grafts, significantly reduced retrograde degeneration and atrophy in the injured red nucleus. Grafts of BDNF + NT-3 expressing fibroblasts delayed 6 weeks after injury therefore elicit growth from intact segmental and descending spinal tracts, stimulate modest regenerative growth by rubrospinal axons, and partially rescue axotomized supraspinal neurons and protect them from atrophy. The regeneration of rubrospinal axons into delayed transplants was much less than has been observed when similar transplants were placed acutely into a lateral funiculus or, after a 4-week delay, into a hemisection lesion. This suggests that the regenerative capacity of chronically injured red nucleus neurons was markedly diminished. The increased GAP43 reactivity in the corticospinal tracts ipsilaterally and contralaterally to the combination grafts suggests that these axons remain responsive to the neurotrophins, that the neurotrophins may stimulate both regenerative and sprouting responses, and that the grafted cells continue to secrete the neurotrophins.
authors.affiliation: Department of Neurobiology and Anatomy, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Tobias
authors.forename: J S
authors.initials: JS
authors.lastname: Shumsky
authors.forename: M
authors.initials: M
authors.lastname: Shibata
authors.forename: M H
authors.initials: MH
authors.lastname: Tuszynski
authors.forename: I
authors.initials: I
authors.lastname: Fischer
authors.forename: A
authors.initials: A
authors.lastname: Tessler
authors.forename: M
authors.initials: M
authors.lastname: Murray
mesh_terms: D000818:Animals; D001284:Atrophy; D019771:Axotomy; D001710:Biotin / Q000031:analogs & derivatives*; D019208:Brain-Derived Neurotrophic Factor / Q000096:biosynthesis*; D002452:Cell Count; D048429:Cell Size; D002470:Cell Survival; D016572:Cyclosporine / Q000494:pharmacology; D003911:Dextrans; D005260:Female; D005347:Fibroblasts / Q000378:metabolism* / Q000637:transplantation*; D019922:GAP-43 Protein / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008164:Luminescent Proteins / Q000096:biosynthesis; D009416:Nerve Regeneration / Q000502:physiology*; D009474:Neurons / Q000473:pathology* / Q000502:physiology; D020933:Neurotrophin 3 / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D012012:Red Nucleus / Q000473:pathology*; D013119:Spinal Cord Injuries / Q000473:pathology / Q000601:surgery* / Q000628:therapy
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D003911:Dextrans; D019922:GAP-43 Protein; D007166:Immunosuppressive Agents; D008164:Luminescent Proteins; D020933:Neurotrophin 3; D011994:Recombinant Proteins; C076397:biotinylated dextran amine; D049452:Green Fluorescent Proteins; D001710:Biotin; D016572:Cyclosporine