Expression profiling of spinal cord dorsal horn in a rat model of complex regional pain syndrome type-I uncovers potential mechanisms mediating pain and neuroinflammation responses

Chen et al. Journal of Neuroinflammation (2020) 17:162 https://doi.org/10.1186/s12974-020-01834-0 RESEARCH Open Access Expression profiling of spinal cord dorsal horn in a rat model of complex regional pain syndrome type-I uncovers potential mechanisms mediating pain and neuroinflammation responses Ruixiang Chen1†, Chengyu Yin1†, Qimiao Hu1†, Boyu Liu1, Yan Tai2, Xiaoli Zheng1, Yuanyuan Li1, Jianqiao Fang1* and Boyi Liu1* Abstract Background: Complex regional pain syndrome type-I (CRPS-I) is a progressive and devastating pain condition. The mechanisms of CRPS-I still remain poorly understood. We aim to explore expression profiles of genes relevant to pain and neuroinflammation mechanisms involved in CRPS-I. Methods: The rat chronic post-ischemic pain (CPIP) model that mimics human CRPS-I was established. RNAsequencing (RNA-Seq), qPCR, Western blot, immunostaining, and pharmacological studies were used for profiling gene changes in ipsilateral spinal cord dorsal horn (SCDH) of CPIP model rat and further validation. Results: CPIP rats developed persistent mechanical allodynia in bilateral hind paws, accompanied with obvious glial activation in SCDH. RNA-Seq identified a total of 435 differentially expressed genes (DEGs) in ipsilateral SCDH of CPIP rats. qPCR confirmed the expression of several representative genes. Functional analysis of DEGs identified that the most significantly enriched biological processes of upregulated genes include inflammatory and innate immune response. We further identified NLRP3 inflammasome expression to be significantly upregulated in SCDH of CPIP rats. Pharmacological blocking NLRP3 inflammasome reduced IL-1β overproduction, glial activation in SCDH as well as mechanical allodynia of CPIP rats. Conclusion: Our study revealed that immune and inflammatory responses are predominant biological events in SCDH of CPIP rats. We further identified NLRP3 inflammasome in SCDH as a key contributor to the pain and inflammation responses in CPIP rats. Thus, our study provided putative novel targets that may help to develop effective therapeutics against CRPS-I. Keywords: RNA-Seq, Pain, CRPS-I, Spinal cord dorsal horn, Inflammation, Inflammasome, Cytokine * Correspondence: ; † Ruixiang Chen, Chengyu Yin and Qimiao Hu contributed equally to this work. 1 Department of Neurobiology and Acupuncture Research, The Third Clinical Medical College, Zhejiang Chinese Medical University, Key Laboratory of Acupuncture and Neurology of Zhejiang Province, 548 Binwen Road, Hangzhou 310053, China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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 in a credit line to the data. Chen et al. Journal of Neuroinflammation (2020) 17:162 Introduction Complex regional pain syndrome type-I (CRPS-I) is a progressive and devastating neuropathic pain condition that usually affects the limb and is not accompanied with a clinically verifiable nerve injury [1]. CRPS-I usually develops after an initial injury, which includes ischemia, soft tissue trauma, surgery, or fractures to the extremity [2, 3]. CRPS-I can develop into chronic pain state that severely affects the patient’s life quality [4]. There are currently no specific drugs approved for the treatment of CRPS. Conventional treatments for CRPS-I include physiotherapy, sympathetic blockade, corticosteroids, and non-steroidal anti-inflammatory drugs (NSAIDs) [5]. However, none of the above treatment options produce satisfactory relieving effects on CRPS-I, which makes it one of the most clinically challenging neuropathic pain conditions [6]. Currently, the mechanisms of CRPS-I are still not fully understood. Ischemia/reperfusion injury is among one of the major causes leading to CRPS-I [7, 8]. In order to get more understandings of the mechanisms of CRPS-I, Coderre et al. established a rat chronic post-ischemic pain (CPIP) model by applying prolonged hind paw ischemia and reperfusion to mimic CRPS-I [9]. The CPIP model displayed many CRPS-I-like symptoms, including early hind paw edema, hyperemia, and skin warmth, accompanied with long-lasting neuropathic pain conditions, including bilateral mechanical and thermal hypersensitivities [9]. With the aid of this animal model, it is proposed that central pain sensitization, reactive oxygen species increase, TRPA1 activation, etc. may contribute to CRPS-I pathology [10–12]. We recently contributed to these efforts by performing transcriptome profiling of the dorsal root ganglia (DRG) of CPIP model rat and identified potential peripheral pain mechanisms involved in CRPS-I [13]. Our recent work further identified the pain-sensing ion channel TRPV1 in the DRG neurons as a key target involved in mediating the pain symptoms of CPIP model rats [14]. The spinal cord dorsal horn (SCDH) receives pain signal inputs from the peripheral sensory neurons and plays a critical role in integrating pain signals and central pain sensitization. Non-neuronal cells, such as astrocytes and microglia, are activated in SCDH of CPIP model rats and produce pro-inflammatory mediators, such as some cytokines and chemokines that can modulate pain process [11, 15, 16]. These substances act on spinal nociceptive neurons to produce neuroinflammation and sensitize pain-related receptors or ion channels to initiate central pain sensitization [17]. In order to further explore the central mechanisms underlying CRPS-I, we proceeded to carry out genome-wide expression profiling of the ipsilateral SCDH of CPIP model rats and sham control rats using RNA-Seq. We identified a number of Page 2 of 19 differentially expressed genes (DEGs). We further examined the molecular/cellular functions and the signaling pathways that these DEGs were involved in. We compared our findings with previously published datasets of neuropathic pain models and identified a core set of genes and pathways that extensively participated in CPIP and other neuropathic pain conditions. By analyz (...truncated)