Differential transcriptional profiling of damaged and intact adjacent dorsal root ganglia neurons in neuropathic pain

PLoS One. 2015 Apr 16;10(4):e0123342. doi: 10.1371/journal.pone.0123342. eCollection 2015.

Abstract

Neuropathic pain, caused by a lesion in the somatosensory system, is a severely impairing mostly chronic disease. While its underlying molecular mechanisms are not thoroughly understood, neuroimmune interactions as well as changes in the pain pathway such as sensitization of nociceptors have been implicated. It has been shown that not only are different cell types involved in generation and maintenance of neuropathic pain, like neurons, immune and glial cells, but, also, intact adjacent neurons are relevant to the process. Here, we describe an experimental approach to discriminate damaged from intact adjacent neurons in the same dorsal root ganglion (DRG) using differential fluorescent neuronal labelling and fluorescence-activated cell sorting (FACS). Two fluorescent tracers, Fluoroemerald (FE) and 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), were used, whose properties allow us to distinguish between damaged and intact neurons. Subsequent sorting permitted transcriptional analysis of both groups. Results and qPCR validation show a strong regulation in damaged neurons versus contralateral controls as well as a moderate regulation in adjacent neurons. Data for damaged neurons reveal an mRNA expression pattern consistent with established upregulated genes like galanin, which supports our approach. Moreover, novel genes were found strongly regulated such as corticotropin-releasing hormone (CRH), providing novel targets for further research. Differential fluorescent neuronal labelling and sorting allows for a clear distinction between primarily damaged neuropathic neurons and "bystanders," thereby facilitating a more detailed understanding of their respective roles in neuropathic processes in the DRG.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carbocyanines / analysis
  • Carbocyanines / metabolism
  • Corticotropin-Releasing Hormone / genetics
  • Dextrans / analysis
  • Dextrans / metabolism
  • Female
  • Flow Cytometry / methods
  • Fluoresceins / analysis
  • Fluoresceins / metabolism
  • Fluorescent Dyes / analysis
  • Fluorescent Dyes / metabolism
  • Ganglia, Spinal / injuries*
  • Ganglia, Spinal / physiopathology
  • Gene Expression Profiling*
  • Mice, Inbred C57BL
  • Neuralgia / genetics*
  • Neuralgia / pathology
  • Neurons / pathology
  • Sensory Receptor Cells / metabolism
  • Sensory Receptor Cells / pathology

Substances

  • 3,3'-dihexadecylindocarbocyanine
  • Carbocyanines
  • Dextrans
  • Fluoresceins
  • Fluorescent Dyes
  • fluoro-emerald
  • Corticotropin-Releasing Hormone

Grants and funding

This study was technically supported by EMBL Monterotondo Flow cytometry facility and EMBL Genomics Core Facility. The study has received funding from the project “ncRNAPain” under the European Union’s 7th framework programme (Grant no. 602133).