Buckley, David A. (2018) Improving the Diagnosis and Treatment of Chronic Neuropathic Pain. Doctoral thesis, University of Huddersfield.

Chronic neuropathic pain (CNP) occurs as a consequence of injury to the nervous system. Despite recent advances, CNP lacks objective diagnostic criteria, is often unrelenting and refractory to treatment. The primary aims of this thesis are twofold; the identification of CNP biomarkers using both human cohorts and an animal model (spinal nerve ligation; SNL) of neuropathic pain, and to provide clarity on the role of GTP cylcohydrolase I (GCH1) in CNP.

Analysis of GCH1 and related genes and metabolites was conducted. As biomarkers, nitrite/nitrate and neopterin did not differentiate controls from CNP patients. However, significant differences were observed with biopterins, whilst correlations were observed between GCH1, nitrite/nitrate and neopterin, which were notably stronger in patients than controls. Analysis in human cohorts and in the SNL model also inferred that downregulation of GCHFR may contribute to BH4 synthesis.

In order to provide clarity on the role of the GCH1 pain protective haplotype, reporter gene assays were used. This demonstrated a potential regulatory role for the GCH1 5’ SNP (rs8007267). In silico prediction of transcription factor binding sites suggested that this may be mediated by the aryl hydrocarbon nuclear translocator. The use of electrophoretic mobility shift assays showed strong specific binding with probe pertaining to the major allele. Further analysis is required to elucidate transcription factor binding, potentially facilitated by 2D-PAGE and mass spectrometry.

In order to further elucidate potential CNP biomarkers, microarray analysis and qRT-PCR were performed using blood obtained from CNP patients. Data refinement led to the isolation of 27 potential CNP biomarkers, of which several cross-validated between cohorts. Microarray data, literature evidence, and correlations with previous microarrays provided evidence suggestive of a role for TIMP1. Multiple other genes, including CASP5, TLR4, TLR5, MC1R and CX3CR1, were differentially regulated in CNP. Genes surviving microarray data refinement were subsequently analysed in the dorsal horn of Sprague Dawley and Wistar Kyoto rats after SNL. Several genes, including Dpp3, Mc1r and Timp1, were similarly differentially expressed in the rodent SNL model, which suggests that these genes may be involved in the pathophysiological mechanisms of CNP, and may also function as potential translational biomarkers of CNP.

This work provides multiple avenues for expansion and further investigation. Clearly, the challenges associated with biomarker discovery in CNP states are considerable, though it is hoped that this thesis provides valuable insight and the necessary foundation for future work.

David Buckley FINAL THESIS.PDF - Accepted Version
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