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

Reinhold AK, Batti L, Bilbao D, Buness A, Rittner HL, Heppenstall PA - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Monterotondo, Italy; Department of Anaesthesiology, University Hospital, Würzburg, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

Flow cytometric detection of damaged and intact neurons for qPCR.DRGs L3-5 were harvested and cells isolated 7 days after CCI. The sorting strategy to identify neurons positive for Fluoroemerald (FE) and DiI is shown in (A). Initially, cells were gated for size and granularity, before excluding dead cells using Sytox Blue and haematopoetic cells using CD45-Ab Cy7. The remaining cells were sorted for DiI and FE. FACS plots of negative control (B left), contralateral (B middle) and ipsilateral (B right) DRG cells are displayed in the lower panel. DiI+/FE- cells are considered to be spared neurons, FE+ cells are damaged neurons. Both populations were obtained for further analysis (n = 3, representative example).
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pone.0123342.g002: Flow cytometric detection of damaged and intact neurons for qPCR.DRGs L3-5 were harvested and cells isolated 7 days after CCI. The sorting strategy to identify neurons positive for Fluoroemerald (FE) and DiI is shown in (A). Initially, cells were gated for size and granularity, before excluding dead cells using Sytox Blue and haematopoetic cells using CD45-Ab Cy7. The remaining cells were sorted for DiI and FE. FACS plots of negative control (B left), contralateral (B middle) and ipsilateral (B right) DRG cells are displayed in the lower panel. DiI+/FE- cells are considered to be spared neurons, FE+ cells are damaged neurons. Both populations were obtained for further analysis (n = 3, representative example).

Mentions: In the next step we analyzed cells in the DRG regarding their uptake of the two markers DiI for intact neurons and FE for damage neurons by flow cytometry before sorting. In the initial flow cytometric analysis of single cell suspensions from mice DRG 7 d after CCI, 2.01% of viable cells included were Dil+/FE- (adjacent intact neurons) and 1.01% of all cells were FE+ (consistent with damaged neurons) (S1 Fig). No FE+ cells were found contralaterally. These sorted cells were further used for the gene expression profile Percentage of DiI+ undamaged neuron detected varied in runs for qPCR. For samples preparation for qPCR, CD45+ hematopoietic cells were also excluded (Fig 2).


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

Reinhold AK, Batti L, Bilbao D, Buness A, Rittner HL, Heppenstall PA - PLoS ONE (2015)

Flow cytometric detection of damaged and intact neurons for qPCR.DRGs L3-5 were harvested and cells isolated 7 days after CCI. The sorting strategy to identify neurons positive for Fluoroemerald (FE) and DiI is shown in (A). Initially, cells were gated for size and granularity, before excluding dead cells using Sytox Blue and haematopoetic cells using CD45-Ab Cy7. The remaining cells were sorted for DiI and FE. FACS plots of negative control (B left), contralateral (B middle) and ipsilateral (B right) DRG cells are displayed in the lower panel. DiI+/FE- cells are considered to be spared neurons, FE+ cells are damaged neurons. Both populations were obtained for further analysis (n = 3, representative example).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400143&req=5

pone.0123342.g002: Flow cytometric detection of damaged and intact neurons for qPCR.DRGs L3-5 were harvested and cells isolated 7 days after CCI. The sorting strategy to identify neurons positive for Fluoroemerald (FE) and DiI is shown in (A). Initially, cells were gated for size and granularity, before excluding dead cells using Sytox Blue and haematopoetic cells using CD45-Ab Cy7. The remaining cells were sorted for DiI and FE. FACS plots of negative control (B left), contralateral (B middle) and ipsilateral (B right) DRG cells are displayed in the lower panel. DiI+/FE- cells are considered to be spared neurons, FE+ cells are damaged neurons. Both populations were obtained for further analysis (n = 3, representative example).
Mentions: In the next step we analyzed cells in the DRG regarding their uptake of the two markers DiI for intact neurons and FE for damage neurons by flow cytometry before sorting. In the initial flow cytometric analysis of single cell suspensions from mice DRG 7 d after CCI, 2.01% of viable cells included were Dil+/FE- (adjacent intact neurons) and 1.01% of all cells were FE+ (consistent with damaged neurons) (S1 Fig). No FE+ cells were found contralaterally. These sorted cells were further used for the gene expression profile Percentage of DiI+ undamaged neuron detected varied in runs for qPCR. For samples preparation for qPCR, CD45+ hematopoietic cells were also excluded (Fig 2).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Monterotondo, Italy; Department of Anaesthesiology, University Hospital, Würzburg, Germany.

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.

No MeSH data available.


Related in: MedlinePlus