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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

Principle of fluorescent tracer injection and DiI signal after CCI.(A) Fluoroemerald (FE, green) is applied proximal to the site of injury; it is taken up by damaged neurons and transported to the DRG. DiI (red) is injected into the hindpaw immediately after the surgical procedure. It permeates the axonal membrane and diffuses along the axon. Membrane disruption, however, impedes further diffusion towards the DRG. (B). One week after CCI, the sciatic nerve was excised and cryosected to examine native DiI intensity. Compared to the distal sciatic nerve (left panel), transections proximal to the site of injury (right panel) exhibit a clearly decreased DiI intensity. Nuclei were visualized with blue DAPI (n = 4, representative sample, scale bar = 40 μm).
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pone.0123342.g001: Principle of fluorescent tracer injection and DiI signal after CCI.(A) Fluoroemerald (FE, green) is applied proximal to the site of injury; it is taken up by damaged neurons and transported to the DRG. DiI (red) is injected into the hindpaw immediately after the surgical procedure. It permeates the axonal membrane and diffuses along the axon. Membrane disruption, however, impedes further diffusion towards the DRG. (B). One week after CCI, the sciatic nerve was excised and cryosected to examine native DiI intensity. Compared to the distal sciatic nerve (left panel), transections proximal to the site of injury (right panel) exhibit a clearly decreased DiI intensity. Nuclei were visualized with blue DAPI (n = 4, representative sample, scale bar = 40 μm).

Mentions: Immediately following CCI ligation, 2 μl of FE (5% in 0.9% saline, Invitrogen, Carlsbad, CA, USA) were injected epineurally into the exposed nerve proximal to the ligation site using a Hamilton syringe with a 32 G needle or micropipette. Great care was taken not to penetrate deeper layers of the nerve. After closure of the wound, 4 μl DiI (10 mg/ml in dimethyl sulfoxide (DMSO), Carlsbad, CA, USA) were injected subcutaneously into the plantar surface of both hindpaws using a 28 G needle (Fig 1A). The site of injection was manually pressed for one minute to facilitate puncture closure and avoid dye leakage.


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)

Principle of fluorescent tracer injection and DiI signal after CCI.(A) Fluoroemerald (FE, green) is applied proximal to the site of injury; it is taken up by damaged neurons and transported to the DRG. DiI (red) is injected into the hindpaw immediately after the surgical procedure. It permeates the axonal membrane and diffuses along the axon. Membrane disruption, however, impedes further diffusion towards the DRG. (B). One week after CCI, the sciatic nerve was excised and cryosected to examine native DiI intensity. Compared to the distal sciatic nerve (left panel), transections proximal to the site of injury (right panel) exhibit a clearly decreased DiI intensity. Nuclei were visualized with blue DAPI (n = 4, representative sample, scale bar = 40 μm).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123342.g001: Principle of fluorescent tracer injection and DiI signal after CCI.(A) Fluoroemerald (FE, green) is applied proximal to the site of injury; it is taken up by damaged neurons and transported to the DRG. DiI (red) is injected into the hindpaw immediately after the surgical procedure. It permeates the axonal membrane and diffuses along the axon. Membrane disruption, however, impedes further diffusion towards the DRG. (B). One week after CCI, the sciatic nerve was excised and cryosected to examine native DiI intensity. Compared to the distal sciatic nerve (left panel), transections proximal to the site of injury (right panel) exhibit a clearly decreased DiI intensity. Nuclei were visualized with blue DAPI (n = 4, representative sample, scale bar = 40 μm).
Mentions: Immediately following CCI ligation, 2 μl of FE (5% in 0.9% saline, Invitrogen, Carlsbad, CA, USA) were injected epineurally into the exposed nerve proximal to the ligation site using a Hamilton syringe with a 32 G needle or micropipette. Great care was taken not to penetrate deeper layers of the nerve. After closure of the wound, 4 μl DiI (10 mg/ml in dimethyl sulfoxide (DMSO), Carlsbad, CA, USA) were injected subcutaneously into the plantar surface of both hindpaws using a 28 G needle (Fig 1A). The site of injection was manually pressed for one minute to facilitate puncture closure and avoid dye leakage.

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