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.
Highlights
Neuropathic pain is defined as pain arising from a lesion within the somatosensory system
By combining differential fluorescent neuronal labelling with fluorescence-activated cell sorting (FACS), we developed an approach that allows the study of neuron-specific expression and enabled us to compare gene expression in damaged and adjacent intact dorsal root ganglion (DRG) neurons after chronic constriction injury (CCI)
That DiI transport in damaged neurons is reduced after CCI and that CCI-induced damage affects considerable portions of the nerve, transections of the sciatic nerve distal and proximal to the lesion were compared for DiI signalling. 7 days after CCI, DiI intensity in the sciatic nerve proximal to the lesion is decreased greatly compared to distal sites
Summary
Female C57/BL6 mice of 6–8 weeks of age were used (Charles River Laboratories, Wilmington, MA, USA). Mice were housed in sawdust cages (4–5 mice per cage, with water and food provided ad libitum). Mice were anesthetized with an intraperitoneal injection of 2.5% tribromoethanol (Sigma Aldrich, St Louis, MO, USA) or isoflurane 1.5 Vol% (Baxter, Deerfield, IL, USA). The sciatic nerve was located and exposed after skin incision. Three friction-knotted loose ligations were tied around the sciatic nerve using 7–0 silk threads and the wound was closed with a metal clip [20]. Sham surgery consisted of anaesthesia and exposure of the nerve but without nerve ligation. Animal experiments were approved by EMBL Monterotondo Animal Committee and comply with Italian legislation Jan 1992, no 116, under licence from the Italian Ministry of Health)
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