RNA Sequencing of Neuropathic Pain in the Anterior Cingulate Cortex after Nerve Injury


 Background: Neuropathic pain is a troublesome pathological condition without suitable treatments. Anterior Cingulate Cortex (ACC) is a core brain region to process pain emotion. In this study, we performed RNA sequencing analysis to reveal transcriptomic profiles of the ACC in a rat chronic constriction injury (CCI) model.Results: A total of 1628 differentially expressed genes (DEGs) were identified by comparing the sham-operated rats and rats of 12 hours, 1, 3, 7 and 14 days after surgery, respectively. Most of the DEGs were involved in inflammatory and immune process. Although these inflammatory-related DEGs were generally increased after CCI, they demonstrated different kinetics in time-series expression with the development of neuropathic pain affection. Specifically, the expression of Ccl5 , Cxcl9 and Cxcl13 were kept going up after CCI, indicating a potentially effect of these genes on initiation and maintenance of neuropathic pain affection. The expression of Ccl2 , Ccl3 , Ccl4 , Ccl6 and Ccl7 were initially upregulated at 12 hours after CCI and then they fell back after that. Similarly, the expression of Rac2 , Cd68 , Icam-1 , Ptprc , Itgb2 , Fcgr2b were rised at 12 hours and 1 day, but fell back at 3 days after CCI. However, the expression of all of the above two clusters of genes were increased again at 7 days after CCI, when the neuropathic pain affection was developed. The initial increase of these genes may indicate an early response of ACC to nerve injury, whereas the later increase of these genes may indicate their involvement in the developing of neuropathic pain affection. Gene Ontology analysis, KEGG pathway enrichment and interaction network analysis further showed a high connectivity degree among these chemokine targeting genes. Similar expressional changes of these genes were also found in the rat spinal dorsal taking charge of the processing of nociception.Conclusions: Our results indicate chemokines and their targeting genes in ACC may be differentially involved in the initiation and maintenance of neuropathic pain affection. These genes could be the target not only the nociception but also the pain affection subsequent to nerve injury.


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Neuropathic pain caused by the injury or disease of somatosensory nervous 14 system is a pathological condition, which brings great trouble to patients. 15 According to the data of the International Association for the study of pain in 16 2019, about 10% of the world's population suffered from neuropathic pain 1 . It is 17 of great significance to deeply study the pathogenesis of neuropathic pain and 18 explore effective treatment drugs for the development of human health. 19 ACC located in the forebrain is an important part of limbic system. 20 Accumulating evidence shows that ACC is a core brain region to process pain 21 emotion. Clinical studies have shown that the structure and function of ACC in 22 patients with chronic pain change significantly 2-4 .The synaptic plasticity is the 1 key mechanism of the occurrence and development of chronic pain in ACC 2 supported by a large number of animal experiments 5,6 . Peripheral inflammatory 3 pain or neuropathic pain can cause significant changes in synaptic 4 transmission and morphological plasticity of ACC neurons 7-9 , and blocking this 5 abnormal plasticity of ACC plays a significant analgesic effect 10,11 . Long-term 6 potentiation (LTP) is a form of synaptic plasticity that have been studied in the 7 context of learning, memory, and chronic pain 5,12,13 . LTP includes two forms, 8 one is presynaptic form of LTP (pre-LTP) and the other is postsynaptic form of 9 LTP (post-LTP) 14 . In ACC, post-LTP works in a glutamatergic 10 NMDA-dependent manner to sustain the pain affection, which involves 11 adenylyl cyclase type 1 (AC1) and a-amino-3-hydroxy-5-methyl-4-isoxazole 12 propionic acid (AMPA) receptor 10 . However, pre-LTP is triggered by kainate 13 receptors, rather than NMDA receptors, involved in anxiety-like behaviors 15 . 14 To date, RNA sequencing analysis about ACC have been conducted to 15 reveal the mechanism underlying psychiatric disorders [16][17][18] , diurnal rhythms 19 16 and cognitive dysfunction 20 , however, with few in neuropathic pain. A recent 17 study revealed a whole transcriptome in the spinal cord, ACC, and amygdala 18 following spinal nerve ligation (SNI). But the comparison was just between 19 sham-surgery and SNI-7days mice 21 . In order to explore a comprehensive their targeting genes in ACC may be differentially involved in the initiation and 6 maintenance of neuropathic pain affection. These genes could be the target 7 not only the nociception but also the pain affection subsequent to nerve injury. We performed CCI in rats and then measured PWMT and PWTL of the rats 12 from 12 hours to 14 days. Compared with the sham rats, the PWMT and PWTL 13 of the CCI rats decreased from 3 days to 14 days ( Fig.1A-B). SPT showed a 14 decrease intake of sucrose in CCI rats ( Fig 1C). CCI rats spent more time in 15 the light area to escape the stimulation in the PEAP test, while sham rats were 16 willing to stay in the dark area ( Fig 1D). These results showed that the 17 neuropathic pain model was successfully established.  20 We performed RNA sequencing analysis to reveal transcriptomic profiles of the 21 ACC in CCI rats. The transcriptome data were generated from the rats of 12 22 hours, 1, 3, 7 and 14 days after CCI and sham-operated rats. Figure 2B 1 showed the quality control of sequencing data of each sample. On average, 2 about 60 million clean reads were collected and a mapping rate of round 3 93~97%. The Q20 ratio were all above 94%. Then we calculated the 4 correlation value between every two samples based on normalized expression 5 results and draw a correlation heat map ( Fig. 2A). 6 Our data revealed that there were 1628 DEGs with the cut-offs of fold  To further understand the specific functions of DEGs in ACC of rats after CCI, 3 we performed GO Analysis to analyze the biological process, cellular 4 component and molecular function of the DEGs. GO Analysis identified the 5 biological processes enriched with "immune system process", "defense 6 response", "regulation of immune system process", "cell adhesion" and 7 "cytokine production", suggesting that strong immune and inflammatory 8 response occurred in ACC after CCI. The cellular components were mainly 9 "cell periphery", "vesicle", "intrinsic component of plasma membrane", "integral 10 component of plasma membrane" and "plasma membrane protein complex" 11 indicating that multiple membrane components involved in neuropathic pain.

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The molecular function terms focused on "protein binding", "signaling receptor 13 binding", "G protein-coupled receptor binding", "antigen binding" and "CCR 14 chemokine receptor binding". The GO analysis revealed that the immune and 15 inflammatory responses were key physiological process on the occurrence 16 and development of neuropathic pain (Fig 4A-C). Then we analyze the DEGs through KEGG pathway enrichment to identify the 20 major signaling pathways involved in the neuropathic pain (Fig 5). Among the 21 top30 pathways, the DEGs were enriched in "TNF signaling pathway", 22 "Phagosome", "NF-kappa B signaling pathway", "Cytokine-cytokine receptor 1 interaction", "Complement and coagulation cascades", "Chemokine signaling 2 pathway", "Cell adhesion molecules (CAMs)" and "Antigen processing and 3 presentation", which similarly indicated that the cytokines and complements 4 participated in neuropathic pain largely. Besides, some pathways concerning 5 infection or immune system diseases were also enriched, for instance, "Viral 6 myocarditis" and "Type Ⅰ diabetes mellitus". These results showed that 7 immune and inflammatory responses were pivotal in ACC after CCI. DEGs were up-regulated to a small peak on 12 hours, fell back in day 1, while 17 the DEGs of profile 27 started to be up-regulated at 12 hours, reached a small 18 peak on day 1, and fell back on day 3. Both of them continued to increase after 19 7 days. In addition, the profile 39 DEGs kept going up (Fig 6).

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To further understand the functions of the DEGs in these 3 profiles, we 21 performed GO analysis to identify biological processes, molecular function and 22 cellular component against them. As Supplementary Fig.1A showed, the 1 enriched biological processes of profile 36 were those representing immune 2 processes, they were "immune response", "response to cytokine", "response to 3 interferon-gamma" and "cellular response to interferon-gamma". The role of 4 chemokines in the enriched molecular function terms were particularly 5 prominent, including "cytokine receptor binding", "cytokine activity", 6 "chemokine receptor binding", "chemokine activity" and "CCR chemokine 7 receptor binding". The enriched cellular components were mainly in the 8 nucleus, such as "nuclear chromosome", "chromosome" and "chromatin". In 9 the profile 27, the enriched biological processes were those representing 10 immune processes, they were "immune system process", "cell activation", 11 "lymphocyte aggregation", "leukocyte aggregation", "mononuclear cell 12 proliferation", "lymphocyte proliferation", "leukocyte proliferation" and "T cell 13 proliferation". The enriched molecular function terms were "protein binding", 14 "enzyme binding", "kinase binding" and "MHC class II protein complex binding".

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The enriched cellular components were extranuclear, they were "membrane", 16 "cell periphery" and "plasma membrane". As for the DEGs in the profile 39 , the 17 biological processes were "immune system process", "immune response", 18 "positive regulation of immune system process" and "cell activation" ， the 19 molecular function terms were "anion binding", "carbohydrate derivative 20 binding" and "ribonucleoside binding", and the cellular components were 21 "protein-containing complex" "plasma membrane protein complex", 22 "chromosome" and "nuclear chromosome". 1 To identify the major signaling pathways involved in the formation and 2 development of neuropathic pain, we continued to analyze DEGs in profile 27、 such as "Fc gamma R-mediated phagocytosis", "B cell signaling pathway", 10 "Leukocyte transendothelial migration" and "T cell receptor signaling pathway".

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These results indicated that the initial increase of these DEGs may indicate an  p-value<1.0e-16. Following genes showed high connectivity degrees: Ccl3(degree=12), Ccl4(degree=11), Ccl7(degree=10), etc.. (Fig 7). These 10 results suggested that chemokine targeting genes in ACC may be differentially 11 involved in the initiation and maintenance of neuropathic pain affection. In order to know the specific expression patterns of each chemokine in ACC, 15 we made a bar chart to show the expression levels of them at different stages. Pain includes pain affection and emotional perception. ACC is a key brain 7 region to regulate pain. In this study, we performed RNA-seq to reveal dynamic 8 transcriptomic profiles of the ACC from CCI rats. We found 1628 DEGs were 9 mainly involved in inflammatory and immune process. Although these Another profile of DEGs also appeared to be up-regulated twice, but the 3 first time was from 12 hours to 1 day after CCI. Compared with DEGs in above 4 profile, the GO analysis and KEGG pathway enrichment of this profile showed 5 that 1) the biological processes about various immune cells activation, 6 proliferation, aggregation and adhesion were particularly prominent. 2) cellular 7 component were "membrane" 、 "cell surface" 、 "external side of plasma 8 membrane" and "cell periphery" rather than mainly in the nucleus. 3)molecular 9 function were "protein binding" 、 "enzyme binding" 、 "MHC class II protein 10 binding" and "MHC class II protein complex binding" . 4) the signaling pathway 11 including "T cell receptor signaling pathway" 、 "Natural killer cell mediated 12 cytotoxicity"、"Leukocyte transendothelial migration"、"Fc gamma R-mediated 13 phagocytosis" and "B cell receptor signaling pathway" were mainly   In conclusion, we found that ACC was not only involved in emotion 2 regulation, but also in connection with inflammatory and immune responses in  the rats were anesthetized with isoflurane, the left sciatic nerve was exposed 22 and tied around by four snug ligatures (4-0) with the same tightness and 1 intervals. After ligation, the nerve was repositioned. In sham surgery rats, the 2 left sciatic nerve was just exposed without ligation.  Sucrose preference test (SPT) was performed according to a previous study 25 . 1 Rats were given 1% sucrose solution for 3-5 days. Then, the experiment was 2 carried out on 1 day before CCI(BL), 3, 7 and 14 days after CCI. During the 3 test, rats were fed in single cage and given two bottles of water (1% sucrose 4 solution and water). The positions of sucrose solution and water were 5 exchanged every 12 hours. After 24 hours, the consumption of sucrose 6 solution and water was measured and the percentage of sucrose preference 7 was calculated.    The GraphPad Prism 7 was used for statistical analysis. Data were analyzed 9 by two-way repeated measures ANOVA followed by Sidak's multiple 10 comparisons test. All data were presented as mean ± SD and statistical 11 significance was set at p < 0.05.   The authors declare that they have no competing interests.                                                represented as means ± SD. Two-way ANOVA; *P < 0.05 vs sham group，***P 6 < 0.001 vs sham group. The summary of quality control of raw RNA-seq data set. It shows Raw reads,

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Clean reads, Clean ratio, Mapping ratio and Q20 (Phred quality scores Q) of 12 18 samples.    The KEGG terms were plotted as the ordinate and the rich factor is plotted as 2 the abscissa. The size of the dots represented the gene number. Behavioral Characterization of The Rats After CCI Mechanical allodynia (A) and thermal hyperalgesia (B)were induced after CCI surgery (n =8). C. Sucrose preference test (n =8). D. CCI rats spent more time in the light area than sham rats in the PEAP test (n =6). Results are represented as means ± SD. Two-way ANOVA; *P < 0.05 vs sham group***P < 0.001 vs sham group.

Figure 2
The quality control of raw RNA-seq data set about ACC A. Heatmap of the correlation between each sample with the Pearson test. B. The summary of quality control of raw RNA-seq data set. It shows Raw reads, Clean reads, Clean ratio, Mapping ratio and Q20 (Phred quality scores Q) of 18 samples.

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