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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

CRH expression in DRG neurons after CCI.(A) mRNA from DRG neurons 7 d after CCI was analyzed by qPCR, It shows a strong, significant upregulation in damaged neurons and, to a smaller degree, in adjacent neurons compared to contralateral neurons (mean ± SEM, n = 2, paired t-statistics *p < 0.05 compared to contralateral). (B) Immunohistochemistry for CRH in DRG neurons after CCI. Ipsilateral DRG were obtained 7 d after CCI or sham surgery and immunostained for CRH. Immunoreactivity for CRH was very low and nearly undetectable in sham controls (right panel). DRGs from neuropathic animals (left panel) show a robust immunoreactivity of CRH, detected in the cytoplasm of small, medium and large size cell bodies (scale bar: 60 αm, two representative samples, n = 3).
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pone.0123342.g006: CRH expression in DRG neurons after CCI.(A) mRNA from DRG neurons 7 d after CCI was analyzed by qPCR, It shows a strong, significant upregulation in damaged neurons and, to a smaller degree, in adjacent neurons compared to contralateral neurons (mean ± SEM, n = 2, paired t-statistics *p < 0.05 compared to contralateral). (B) Immunohistochemistry for CRH in DRG neurons after CCI. Ipsilateral DRG were obtained 7 d after CCI or sham surgery and immunostained for CRH. Immunoreactivity for CRH was very low and nearly undetectable in sham controls (right panel). DRGs from neuropathic animals (left panel) show a robust immunoreactivity of CRH, detected in the cytoplasm of small, medium and large size cell bodies (scale bar: 60 αm, two representative samples, n = 3).

Mentions: A vast number of expressed genes were significantly regulated in damaged primary sensory neurons compared to contralateral neurons (Tables 2 and 3 and S2 Table). Microarray showed considerable regulation for several ion channels in damaged neurons: Purinergic receptor P2rx3, an adenosine triphosphate(ATP)-gated ion channel was highly upregulated in damaged neurons: 8.2-fold compared to contralateral neurons (p<0.05) and 4.3-fold compared to adjacent neurons (p = 0.09). Other channels with a higher expression compared to contralateral neurons include voltage-gated calcium channel alpha 2 delta subunit 1 (Cacnα2δ1, 3.8-fold, p<0.05), and anoctamin 4, a calcium-activated chloride channel of the TMEM16 family (4-fold, p<0.05). For P2rx3 as well as for cation transport regulator-like 1 (Chac1, Fig 5A and 5B), this observation was validated by qPCR. Downregulated channels include potassium channels Kcnk2 (4-fold), Kcnj10 (5.3-fold), Kcnh8 (5.7-fold), and Kcnn4 (6.6-fold, all p<0.05). Of the classical signalling neuropeptides known to be involved in neuropathic pain, galanin expression was considerably augmented in damaged neurons (7.2-fold vs. contralateral neurons, 3.7-fold vs. adjacent neurons, p<0.05), as well as its receptor Gpr151 (14-fold vs. contralateral, p<0.05, 7.8-fold vs. adjacent, p = 0.07, Fig 5C and 5D). Both results were validated through PCR which also confirmed upregulation of neurotensin, neuropeptide Y, cholecystokinin receptor B and transcription factor Atf3 (Fig 5E–5H). Neuropeptides with a decreased expression in qPCR (though not significant in the microarray analysis) included calcitonin gene-related peptide (Cgrp) and substance P/tachykinin (Tac1, Fig 5I and 5J). The most-upregulated gene was corticotropin releasing hormone (Crh). Its striking overexpression in damaged neurons (<200-fold vs. contralateral, p = 0.06) was also validated by qPCR (Fig 6A). Similarly, expression levels were elevated for kainate receptor-modulator neuropilin and tolloid-like 1 (Neto1, 19-fold vs. contralateral, p = 0.08), syndecan 1 (10-fold vs. contralateral, p = 0.1), AMPAR-associated Shisa9 (14.2-fold vs. contralateral, p<0.05, 11.7-fold vs. adjacent, p = 0.05), SRY box-containing gene 11 (Sox11) (10.7-fold vs. contralateral, p<0.05, 10.5-fold vs. adjacent, p = 0.05), and peptidase inhibitor Serpinb1a (17-fold vs. contralateral, p<0.05, 11.7-fold vs. adjacent, p = 0.08), all of which were confirmed by qPCR (Fig 7A and 7E). Prominently downregulated were genes encoding for water channel aquaporin 4 (-27.1-fold vs. contralateral, p<0.05, -18.3-fold vs. adjacent, p = 0.06) and Ripk4 (-27,3-fold vs. contralateral, p<0.05, Fig 7F and 7G).


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)

CRH expression in DRG neurons after CCI.(A) mRNA from DRG neurons 7 d after CCI was analyzed by qPCR, It shows a strong, significant upregulation in damaged neurons and, to a smaller degree, in adjacent neurons compared to contralateral neurons (mean ± SEM, n = 2, paired t-statistics *p < 0.05 compared to contralateral). (B) Immunohistochemistry for CRH in DRG neurons after CCI. Ipsilateral DRG were obtained 7 d after CCI or sham surgery and immunostained for CRH. Immunoreactivity for CRH was very low and nearly undetectable in sham controls (right panel). DRGs from neuropathic animals (left panel) show a robust immunoreactivity of CRH, detected in the cytoplasm of small, medium and large size cell bodies (scale bar: 60 αm, two representative samples, n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123342.g006: CRH expression in DRG neurons after CCI.(A) mRNA from DRG neurons 7 d after CCI was analyzed by qPCR, It shows a strong, significant upregulation in damaged neurons and, to a smaller degree, in adjacent neurons compared to contralateral neurons (mean ± SEM, n = 2, paired t-statistics *p < 0.05 compared to contralateral). (B) Immunohistochemistry for CRH in DRG neurons after CCI. Ipsilateral DRG were obtained 7 d after CCI or sham surgery and immunostained for CRH. Immunoreactivity for CRH was very low and nearly undetectable in sham controls (right panel). DRGs from neuropathic animals (left panel) show a robust immunoreactivity of CRH, detected in the cytoplasm of small, medium and large size cell bodies (scale bar: 60 αm, two representative samples, n = 3).
Mentions: A vast number of expressed genes were significantly regulated in damaged primary sensory neurons compared to contralateral neurons (Tables 2 and 3 and S2 Table). Microarray showed considerable regulation for several ion channels in damaged neurons: Purinergic receptor P2rx3, an adenosine triphosphate(ATP)-gated ion channel was highly upregulated in damaged neurons: 8.2-fold compared to contralateral neurons (p<0.05) and 4.3-fold compared to adjacent neurons (p = 0.09). Other channels with a higher expression compared to contralateral neurons include voltage-gated calcium channel alpha 2 delta subunit 1 (Cacnα2δ1, 3.8-fold, p<0.05), and anoctamin 4, a calcium-activated chloride channel of the TMEM16 family (4-fold, p<0.05). For P2rx3 as well as for cation transport regulator-like 1 (Chac1, Fig 5A and 5B), this observation was validated by qPCR. Downregulated channels include potassium channels Kcnk2 (4-fold), Kcnj10 (5.3-fold), Kcnh8 (5.7-fold), and Kcnn4 (6.6-fold, all p<0.05). Of the classical signalling neuropeptides known to be involved in neuropathic pain, galanin expression was considerably augmented in damaged neurons (7.2-fold vs. contralateral neurons, 3.7-fold vs. adjacent neurons, p<0.05), as well as its receptor Gpr151 (14-fold vs. contralateral, p<0.05, 7.8-fold vs. adjacent, p = 0.07, Fig 5C and 5D). Both results were validated through PCR which also confirmed upregulation of neurotensin, neuropeptide Y, cholecystokinin receptor B and transcription factor Atf3 (Fig 5E–5H). Neuropeptides with a decreased expression in qPCR (though not significant in the microarray analysis) included calcitonin gene-related peptide (Cgrp) and substance P/tachykinin (Tac1, Fig 5I and 5J). The most-upregulated gene was corticotropin releasing hormone (Crh). Its striking overexpression in damaged neurons (<200-fold vs. contralateral, p = 0.06) was also validated by qPCR (Fig 6A). Similarly, expression levels were elevated for kainate receptor-modulator neuropilin and tolloid-like 1 (Neto1, 19-fold vs. contralateral, p = 0.08), syndecan 1 (10-fold vs. contralateral, p = 0.1), AMPAR-associated Shisa9 (14.2-fold vs. contralateral, p<0.05, 11.7-fold vs. adjacent, p = 0.05), SRY box-containing gene 11 (Sox11) (10.7-fold vs. contralateral, p<0.05, 10.5-fold vs. adjacent, p = 0.05), and peptidase inhibitor Serpinb1a (17-fold vs. contralateral, p<0.05, 11.7-fold vs. adjacent, p = 0.08), all of which were confirmed by qPCR (Fig 7A and 7E). Prominently downregulated were genes encoding for water channel aquaporin 4 (-27.1-fold vs. contralateral, p<0.05, -18.3-fold vs. adjacent, p = 0.06) and Ripk4 (-27,3-fold vs. contralateral, p<0.05, Fig 7F and 7G).

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