Intrathecal injection of fluorocitric acid inhibits the activation of glial cells causing reduced mirror pain in rats

Cao et al. BMC Anesthesiology 2014, 14:119 http://www.biomedcentral.com/1471-2253/14/119 RESEARCH ARTICLE Open Access Intrathecal injection of fluorocitric acid inhibits the activation of glial cells causing reduced mirror pain in rats Jing Cao*, Zhihua Li, Zhenhua Zhang, Xiuhua Ren, Qingzan Zhao, Jinping Shao, Ming Li, Jiannan Wang, Puchao Huang and Weidong Zang* Abstract Background: Growing evidence has shown that unilateral nerve injury results in pain hypersensitivity in the ipsilateral and contralateral sides respective to the injury site. This phenomenon is known as mirror image pain (MIP). Glial cells have been indicated in the mechanism of MIP; however, it is not clear how glial cells are involved in MIP. Methods: To observe phenomenon MIP and the following mechanism, 20 adult male Sprague–Dawley rats (weighing 180–220 g) were separated into two groups: Sham Group (n = 10) and left L5 spinal nerve ligated and sectioned (SNL) group (n = 10). Thermal hyperalgesia and mechanical hypersensitivity were measured for both groups to determine if the SNL model had Mirror image of Pain (MIP). Nav1.7 protein expression in DRG was analyzed using immunohistochemistry and western-blotting. And then to observe the effect of fluorocitrate on MIP, 15 rats were separated into three Groups: Sham Group (n = 5); SNL + FC group: intrathecal injection of Fluorocitric acid (FC) 1 nmol/10 μL (n = 5); SNL + NS group: intrathecal injection of 0.9% Normal Saline (n = 5). Behavior testing, immunocytochemistry, and western-blotting using dorsal root ganglion (DRG) from both sides were then conducted. Results: The results showed pain hypersensitivity in both hind-paws of the SNL animals, Mechanical tests showed the paw withdrawal threshold dropped from 13.30 ± 1.204 g to 2.57 ± 1.963 g at 14 d as will as the ipsilateral paw thermal withdrawal threshold dropped from 16.5 ± 2.236 s to 4.38 ± 2.544 s at 14d. Mechanical tests showed the contralateral paw withdrawal threshold dropped from 14.01 ± 1.412 to 4.2 ± 1.789 g at 7d will the thermal withdrawal threshold dropped from 16.8 ± 2.176 s to 7.6 ± 1.517 s at 7d. Nav1.7 expression increased and glial cells actived in bilateral side DRG after SNL compared with sham group. After intrathecal injection of fluorocitrate, the glial cell in bilatral DRG were inhibited and the pain behavior were reversed in both hindpaws too. Conclusions: Fluorocitrate can inhibit the activation of glial cells in spinal cord and DRG, and reduce MIP. Keywords: Mirror-image pain, Satellite glial cells, DL-fluorocitric acid, Nav1.7 protein Background A growing body of evidence indicates that unilateral nerve injury results in bilateral cellular and molecular changes in the nerve structure and pain sensitivity [1,2]. This phenomenon is known as MIP [3]. To date, the mechanism of MIP is still unclear. Deepak Behera [4] used Manganese-enhanced magnetic resonance imaging (MRI) to show increased manganese uptake in both the injured * Correspondence: ; Department of Basic Medical Sciences, Laboratory of Anatomy, Zhengzhou University, Zhengzhou 450001, Henan Province, China sciatic nerve and contralateral sciatic nerve in the chronic constriction injury model of neuropathic pain. Although poorly understood, this finding corroborates the ex vivo finding of bilateral nociceptive-related molecular changes in the nervous system of unilateral pain models. It may be related to humoral immunity, central sensitization, and/or cortical downstream regulation. Surprisingly, evidence of changes in primary neurons and satellite glial cells (SGCs) in regards to MIP is lacking. Because of their unique location in sensory and autonomic ganglion, SGCs can strongly influence nociceptive sensation [5]. © 2014 Cao et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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. Cao et al. BMC Anesthesiology 2014, 14:119 http://www.biomedcentral.com/1471-2253/14/119 In our preliminary studies we found SCN9A abnormality highly expressed in both bilateral spinal ganglion which correlates with the development of MIP. High expression of Nav1.7 protein in the contralateral side may explain the increase in neuronal in the mirror side. SCN9A encodes a subunit of the voltage-gated channel Nav1.7, in which a single-gene mutation is closely related to a congenital abnormality in which the sensation of pain is lost [6]. Yang yong [7] reported a gain-of function mutation of SCN9A causes erythema acrodynia, a disease of severe episodic pain. Nav1.7 may be a promising candidate for the cause of MIP, but the exact mechanism of its upregulation and the associated increase in neuronal excitability is still unkown. It is possible that SGCs in the contralateral DRG may play a role in primary neuronal sensitization [8,9]. SGCs are found in the peripheral nervous system, particularly in DRG. SGCs are the main glial cells in DRG, and they become activated and proliferate after nerve injury or inflammation [10]. SGCs are arranged in a layer, normally around the neurons to form a complete scabbard film. The SGCs also release substances after nerve injury, which can directly affect the neurons that the SCGs surround [11]. Based on the close proximity of the SGCs and their ability to affect primary neurons, we hypothesize that SGC activation in the contralateral DRG following unilateral peripheral nerve injury leads to increased excitability of contralateral DRG neurons and thus, MIP. To address this hypothesis, a rat MIP model established by nerve distal ligation and section (SNL) was used to identify changes in Nav1.7 expression and SGCs activation. Molecular techniques including RT-PCR, western-blotting, and immunohistochemistry were used to identify changes in the expression of Nav1.7 in DRG. Behavioral tests were also utilized to measure pain hypersensitivity. DL-Fluorocitric acid was used to inhibit SGCs activation, and verify the role of SGCs in Nav1.7 overexpression and pain hypersensitivity. Methods Animals and surgical procedures Adult male Sprague–Dawley rats (6-8 W) of clean grade, weighing 180–220 g(n = 25), were provided by the Experimental Animal Center of Henan Province (license No. SYXK2005-0012). The rats were housed with a 12hour light–dark cycle and free access to food and water. They were kept for 1 week under these conditions before surgery. All procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China [12]. To produce persistent neuropathic pain, SNL was pe (...truncated)