Expression changes of microRNA-1 and its targets Connexin 43 and brain-derived neurotrophic factor in the peripheral nervous system of chronic neuropathic rats

Neumann et al. Mol Pain (2015) 11:39 DOI 10.1186/s12990-015-0045-y Open Access RESEARCH Expression changes of microRNA‑1 and its targets Connexin 43 and brain‑derived neurotrophic factor in the peripheral nervous system of chronic neuropathic rats Elena Neumann1†, Henning Hermanns2†, Franziska Barthel1, Robert Werdehausen1 and Timo Brandenburger1* Abstract Background: MicroRNAs (miRNAs) are involved in the neuroplastic changes which induce and maintain neuropathic pain. However, it is unknown whether nerve injury leads to altered miRNA expression and modulation of pain relevant target gene expression within peripheral nerves. In the present study, expression profiles of miR-1 and the pain-relevant targets, brain derived neurotrophic factor (BDNF) and Connexin 43 (Cx43), were studied in peripheral neuropathic pain, which was induced by chronic constriction injury (CCI) of the sciatic nerve in rats. The expression of miR-1 was investigated in the sciatic nerve, dorsal root ganglion (DRG) and the ipsilateral spinal cord by qPCR. Changes of BDNF and Cx43 expression patterns were studied using qPCR, Western blot analysis, ELISA and immunohistochemistry. Results: In sciatic nerves of naïve rats, expression levels of miR-1 were more than twice as high as in DRG and spinal cord. In neuropathic rats, CCI lead to a time-dependent downregulation of miR-1 in the sciatic nerve but not in DRG and spinal cord. Likewise, protein expression of the miR-1 targets BDNF and Cx43 was upregulated in the sciatic nerve and DRG after CCI. Immunohistochemical staining revealed an endoneural abundancy of Cx43 in injured sciatic nerves which was absent after Sham operation. Conclusions: This study demonstrates that CCI leads to a regulation of miRNAs (miR-1) in the peripheral nervous system. This regulation is associated with alterations in the expression and localization of the miR-1 dependent pain-relevant proteins BDNF and Cx43. Further studies will have to explore the function of miRNAs in the context of neuropathic pain in the peripheral nervous system. Keywords: microRNA, miR-1, Connexin 43 (Cx43), BDNF, Neuropathic pain, Chronic constriction injury (CCI) Background Neuropathic pain is caused by a lesion or disease of the somatosensory system involving alterations in the peripheral and the central nervous system [1]. The exact molecular mechanisms of neuropathic pain are incompletely understood and elucidation of these mechanisms is crucial for the development of new *Correspondence: ‑duesseldorf.de † Elena Neumann and Henning Hermanns contributed equally 1 Department of Anesthesiology, Medical Faculty, Heinrich-HeineUniversity Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany Full list of author information is available at the end of the article mechanism-oriented treatment strategies [2]. Neuroplastic changes in the peripheral and central nervous system, particularly alterations in protein expression in the pain processing neuronal network play a key role in the development of pathological pain [3]. MicroRNAs (miRNAs) are small non-coding RNAs which negatively regulate gene expression at the posttranscriptional level and have significant impact on numerous physiological and pathophysiological cellular processes [4]. MiRNA-targeting molecules are considered as possible future therapeutics for a variety of human diseases [5]. The fact that more than 60% of © 2015 Neumann et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Neumann et al. Mol Pain (2015) 11:39 all human protein-coding genes are putative targets of miRNAs [6] suggests that miRNAs are correspondingly involved in the expression changes in chronic pain states. Increasing evidence suggests a significant role of noncoding RNAs, especially miRNAs, in the pathophysiology and potential treatment options of chronic pain [7]. A possible role of miRNAs in the development of chronic pain has to date been investigated in dorsal root ganglia, the spinal cord or in supraspinal organs [8]. One of the miRNAs being involved in neuropathic pain is miR-1. This miRNA has been shown to be involved in the induction of neuropathic pain [9]. Additionally, miR-1 interacts with the two highly pain-relevant proteins Cx43 and BDNF [10, 11]. It is well known that a multitude of neuroplastic alterations also occur in the peripheral nerve [12]. Furthermore, miRNAs are well abundantly expressed in the peripheral nerve, e.g. in Schwann cells but also in dendrites and axons, where they have been shown to be significantly regulated in response to peripheral nerve injury [13]. However, to date there is not data exploring the expression of miRNAs and pain relevant miRNA-target proteins in the context of neuropathic pain in peripheral nerves. In this study we show that miR-1 is well expressed in sciatic nerves of rats. Furthermore we show that constriction injury of the sciatic nerve leads to a time dependent downregulation of miR-1 in injured nerves. This is accompanied by an upregulated protein expression of Connexin 43 (Cx43) and brain derived neurotrophic factor (BDNF) which are well established miR-1 targets. Results Development of neuropathic pain in rats Mechanical allodynia developed within 6 days after nerve ligation. In the left, injured hind paw, the withdrawal threshold in response to stimulation with von Frey hairs was not altered 4 h (Sham 49.6 ± 0.6 g, CCI 47.3 ± 5.1 g, p = 0.26) and 24 h (Sham 50.0 ± 0.1 g, CCI 45.3 ± 4.6 g, p = 0.06) after CCI. On day 6 (Sham 47.7 ± 1.8 g, CCI 29.3 ± 5.1 g, p = 0.0008) and day 12 (Sham 44.5 ± 6.3 g, CCI 21.9 ± 8.1 g, p = 0.0007) post CCI, the withdrawal thresholds were significantly reduced when compared to Sham-operated animals (Figure 1). Expression of miR‑1 and Cx43 and BDNF messenger RNA The relative expression of miR-1 in sciatic nerve was compared to miR-1 expression in DRG and ipsilateral spinal cord of naïve rats. Expression level of miR-1 was higher in sciatic nerves than in DRG (relative expression DRG vs. nerve 0.37, p < 0.05) and the spinal cord (relative expression spinal cord vs. nerve 0.28, p < 0.05, Figure 2). Page 2 of 9 Figure 1 In vivo data on mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve. Paw withdrawal threshold of Sham (left) and CCI (right) animals at four different time points (left injured paw). Significant allodynia is observed on day 6 and 12 post CCI surgery. Mean ± SD, ***p < 0.001 vs. Sham. Figure 2 qPCR dat (...truncated)