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Investigating molecular mechanisms of neuronal regeneration: a microarray approach.

Blain, Alison Margaret (2009) Investigating molecular mechanisms of neuronal regeneration: a microarray approach. PhD thesis, University of Glasgow.

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Abstract

Injury to the peripheral nervous system (PNS) stimulates a finely regulated regenerative response that generally leads to some recovery of function. In contrast, the response to injury in the adult mammalian central nervous system (CNS) is abortive and adult CNS neurons do not normally regenerate. We used a microarray approach to identify putative regeneration-associated changes in gene expression in the L4 dorsal root ganglion (DRG) in rat models of PNS and CNS injury. Our models included crush injury to both branches of the bifurcating axon of sensory neurons with cell bodies in the DRG (DRGNs). Injury to the peripheral branch at the level of the spinal nerve (SN) results in axonal regeneration and reinnervation. Crush injury of the central branch in the dorsal root (DR) results in active regeneration up to the point of CNS entry at the DR entry zone (DREZ) and subsequent arrest of further growth, while transection injury within the CNS at the level of the dorsal columns (DC) results in abortive and unsuccessful regeneration attempts. These DRGN injury models therefore allowed us to compare the gene expression programmes elicited during active, arrested and abortive regeneration. Following a pilot microarray experiment to optimize experimental parameters and tract tracing and electrophysiological experiments to confirm time points for harvest of DRGs after DR and SN injury, respectively, male Sprague-Dawley rats underwent an L4 SN crush, an L4 DR crush or a bilateral DC transection at the L3/L4 spinal segment boundary. L4 DRGs were collected at 2 weeks (active regeneration) and 6 weeks (arrested regeneration) after DR crush. DRGs were harvested at 6 weeks after SN crush and 2 weeks after DC transection. DRGs harvested from naïve rats served as a control group. Microarray analysis (Affymetrix Rat genome 230 2.0 array) identified several hundred genes showing differential expression (5% FDR) in comparisons of regenerating with non-regenerating conditions. Selected genes were chosen for validation by qRT-PCR. These genes could represent putative regeneration-associated genes and may suggest novel therapeutic interventions to encourage regeneration of the spinal cord following injury. Additionally, we have identified genes upregulated in the DR active regeneration state relative to DR arrested state, which have relevance to root avulsion injury and may provide insight into the mechanisms that prevent regeneration of DR axons through the DREZ to re-enter the spinal cord. We also present evidence that a transcriptional programme consistent with regeneration is mounted within the DRG following DC transection. This lends support to the idea that CNS neurons have intrinsic regenerative capability and that manipulations of the CNS environment may be sufficient to permit regeneration of CNS axons.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: neuronal regeneration, injury models, dorsal root ganglion, microarray, central nervous system, dorsal root, spinal nerve, sciatic nerve, dorsal columns
Subjects: R Medicine > RC Internal medicine > RC0321 Neuroscience. Biological psychiatry. Neuropsychiatry
Q Science > QH Natural history > QH426 Genetics
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology > Molecular Cell and Systems Biology
College of Medical Veterinary and Life Sciences > Institute of Neuroscience and Psychology
Supervisor's Name: Bailey, Dr. Mark and Riddell, Dr. John
Date of Award: 2009
Depositing User: Miss Alison M Blain
Unique ID: glathesis:2009-703
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 23 Apr 2009
Last Modified: 10 Dec 2012 13:24
URI: http://theses.gla.ac.uk/id/eprint/703

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