Matrix-mediated gene transfer to brain cortex and dorsal root ganglion neurones by retrograde axonal transport after dorsal column lesion.
BACKGROUND
In previous studies, we showed that the immobilisation of DNAs encoding basic fibroblast growth factor, neurotrophin-3 and brain-derived neurotrophic factor in a gene-activated matrix (GAM) promotes sustained survival of axotomised retinal ganglion cells after optic nerve injury. Here, we evaluated if the immobilisation of DNAs in a GAM could be an effective approach to deliver genes to axotomised dorsal root ganglion (DRG) neurones after spinal cord injury and if the matrix component of the GAM would modulate the deposition of a dense scar at the injury site.


METHODS
We evaluated the expression of the thymidine kinase (TK) reporter gene in brain cortex and DRG after a bilateral T8 dorsal column (DC) lesion using PCR, RT-PCR and in situ hybridisation analyses. Collagen-based GAMs were implanted at the lesion site and the cellular response to the GAM was assessed using cell-specific markers.


RESULTS
At 1 week post-injury, PCR analyses confirmed that DNATK was retrogradely transported from the DC lesion where the GAM was implanted to the brain cortex and to caudal DRG neurones, and RT-PCR analyses showed expression of mRNATK. At 7 weeks post-injury, DNATK was still be detected in the GAM and DRG. In situ hybridisation localised DNATK and mRNATK within fibroblasts, glia, endothelial and inflammatory cells invading the GAM and in DRG neurones. Interestingly, the presence of a GAM also reduced secondary cavitation and scar deposition at the lesion site.


CONCLUSIONS
These results establish that GAMs act as bridging scaffolds in DC lesions limiting cavitation and scarring and delivering genes both locally to injury-reactive cells and distally to the cerebral cortex and to DRG neuronal somata through retrograde axonal transport.
Gonzalez
Ana Maria
AM
Molecular Neuroscience Group, Division of Medical Sciences, University of Birmingham, Birmingham B15 2TT, UK. a.m.gonzalez.1@bham.ac.uk
Berry
Martin
M
Greenlees
Lydia
L
Logan
Ann
A
Baird
Andrew
A
D000818:Animals; D001370:Axonal Transport; D002540:Cerebral Cortex / Q000378:metabolism*; D004274:DNA, Recombinant / Q000008:administration & dosage* / Q000493:pharmacokinetics*; D005260:Female; D005727:Ganglia, Spinal / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D017403:In Situ Hybridization; D009474:Neurons / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013119:Spinal Cord Injuries / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism
D004274:DNA, Recombinant; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013937:Thymidine Kinase