Nerve damage is usually caused by trauma, disease, or surgical intervention. Inflammatory responses and pain because of nerve damage can cause substantial distress in patients. Damage to the peripheral nervous system can induce inflammatory responses in the central nervous system and neuropathic pain in other nerves 1. Chronic constriction injury (CCI) of the sciatic nerve, developed by Bennett and Xie 2, is a traditional model for studying neuropathic pain 3. Immune cells infiltrate the dorsal root ganglia (DRG) and the spinal cord (SC) after CCI of the rat sciatic nerve 4. Immune and glial cells release inflammatory mediators 5, such as tumour necrosis factor (TNF)-α 6 and interleukin 6 7. The nuclear factor (NF)-κB pathway is also activated, causing persistent inflammatory responses 8 or neuropathic pain 9.
Using the CCI model, scientists have validated treatment effects of different drugs on neuropathic pain 10,11. However, the specific roles of many molecules in neuropathic pain, such as receptor-interacting protein (RIP)1 and RIP3, still require elucidation. RIP1 is a regulator of cellular stress and inflammatory responses 12,13, but its involvement in CCI is poorly understood. A study reported that blockade of RIP1 signalling by a cytomegalovirus RIP1 inhibited inflammatory reactions. Moreover, RIP1 was established as an essential component of the TNF receptor 1 signalling pathway, which mediates the activation of NF-κB, mitogen-activated protein kinases and programmed cell death 14. RIP3 is an apoptosis-inducing kinase 15 and regulates inflammation 16. RIP1 usually forms a complex with RIP3. The RIP1–RIP3 complex regulates programmed necrosis and virus-induced inflammation 17. The RIP1–RIP3 complex promotes inflammatory responses through activation of the NLRP3, which requires the participation of dynamin-related protein 1 (DRP1) 18,19. However, it is unclear whether RIP1 and RIP3 exert proinflammatory or pro-necrosis effects in CCI of the sciatic nerve. In this study, we reproduced the sciatic nerve CCI model and investigated the roles of RIP1 and RIP3 in inflammation and nerve injury. Studies have shown that peripheral nervous system damage causes pathological changes in the DRG, SC and hippocampus (HIP) 20–22. Also, the number of inflammatory cells at the injury site of the sciatic nerve and DRGs increased significantly 14 days after CCI 23. Moreover, CCI leads to marked alterations in the SC 24, as well as in HIP 25. Therefore, we focused on the expression of RIP1 and RIP3 in the DRG, SC and HIP.
A total of 30 C57BL/6 mice (male, 5–6 weeks, 19–24 g) were purchased from the Chongqing Tengxin Biological Technology Co., Ltd and housed under approved conditions with 12/12-h light/dark cycles. All mice had ad libitum access to food and water. The experimental protocols were approved by the Experimental Animal Care and Use Committee of Shanghai No. 6 People’s Hospital (registration number: 2016-0116).
Induction of the chronic constriction injury model
Thirty mice were allocated randomly to sham or CCI groups. Mice in the CCI group were subjected to CCI surgeries as described previously 2. In brief, after an intraperitoneal injection of 7% chloral hydrate (5 mg/kg), the right sciatic nerve was bluntly dissected using a surgical suture line (8-0) and was loosely ligated at four different segments. After observing slight contraction of the right posterior limbs, the muscles and skin were closed with sutures. Mice in the sham group were also subjected to blunt dissection of the right sciatic nerve, but without ligation. Three mice in each group were killed on both the second and the eighth day for sampling. Other mice were killed 14 days after surgeries.
Von Frey filaments test
The paw withdrawal threshold (PWT) was tested by a complete set of 20 Von Frey filaments (Stoelting, Dale Wood, Illinois, USA) every 2 days after the surgeries. In the mouse tests, filaments numbered 2 through 15 were used. The PWT of every mouse was calculated according to the following equation: PWT=10(F ×x +B), where F is the PWT calculated in terms of filament number, x=0.224 and B=−4.00 26.
Enzyme-linked immunosorbent assay
After the mice were killed, the CRG, SC and HIP were harvested. The TNF-α and interferon (IFN)-γ levels in these tissues were examined using the ELISA kit (Elabscience, Texas, USA) according to the manufacturer’s instructions. In brief, 10 mg tissue samples and 10 ml PBS were ground mechanically into a homogenate at 4°C. Supernatants were collected after 10 min, centrifuged at 5000 rpm and 10 μl of each supernatant sample was used for detection. After incubation with biotinylated antibody (100 μl/well) for 1 h at 37°C, the plates were washed five times in PBS and incubated with enzyme substrate for 30 min at 37°C and a chromogenic substrate 3,3′,5,5′-tetramethylbenzidine solution for 15 min at 37°C in the dark. The reaction was then terminated. The optical density was read using a Microplate Reader (Thermo, Waltham, Massachusetts, USA) at 450 nm.
Total RNA was isolated from DRG, SC and HIP with Trizol (Invitrogen, California, USA) according to the manufacturer’s instructions. Random RNA-specific primers were used to reverse transcribe 1 μg of RNA to cDNA using the Bestar qPCR RT Kit (DBI). Gene expression levels were analysed using the Agilent Stratagene Mx3000P PCR machine. RT-PCR primers for RIP1: 5′-GCA CCA GCT GTC AGG GCC AG-3′ and 5′-GCC CAG CTT TCG GGC ACA GT-3′, for RIP3: 5′-TTT GGC CTG TCC ACA TTT CAG-3′ and 5′-GGT TGG CAA CTC AAC TTC TCT T-3′, for PD1: 5′-CCG CTC GAG CTC ACC ATG TGG GTC CGG CAG GTA CCC TGG-3′ and 5′-AGA TCT TCC TCC TCC TCC TTG AAA CCG GCC TTC TGG TTT GGG-3′, for TNF-α: 5′-CCC ACT CTG ACC CCT TTA CT-3′ and 5′-TTT GAG TCC TTG ATG GTG GT-3, for IFN-γ: 5′-AGC AAC AGC AAG GCG AAA AA-3′ and 5′-AGC TCA TTG AAT GCT TGG CG-3′, and for GAPDH: 5′-ATG ACA TCAA GAA GGT GGT G-3′ and 5′-CAT ACC AGG AAA TGA GCT TG-3′. The relative expression was analysed using the
Tissues of the DRG, SC and HIP were lysed with lysis buffer (Cell Signaling Technology, Danvers, Massachusetts, USA), and the concentrations were detected using a BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, Illinois, USA). Total proteins (30 μg) were separated and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, Massachusetts, USA). Membranes were blocked with 5% nonfat dry milk for 1 h and incubated with the primary antibodies overnight. The membranes were incubated with the following primary antibodies: anti-RIP1 (1 : 1000 dilution; Cell Signaling Technology), anti-RIP3 (1 : 1000 dilution; Cell Signaling Technology), anti-PD1 (1 : 1000 dilution; Abcam, Cambridge, Massachusetts, USA) and anti-β-actin (1 : 1000 dilution; Abcam) overnight at 4°C. After incubation with the secondary antibody, the results were detected using an ECL chemiluminescence kit (Beyotime, Shanghai, China).
Statistical analysis was carried out using GraphPad Prism 5 (GraphPad, San Diego, California, USA). Group differences in behaviour results and serum cytokines at different time points were analysed by repeated-measures two-way analysis of variance (group×time). Individual protein expression and mRNA were analysed using Student’s t-test. Differences were considered significant when P less than 0.05.
Chronic constriction injury promoted neuroinflammatory responses
The PWT was not significantly different from before surgeries (0 day) to the first day after surgeries, whereas a significant decline was observed in the CCI group at later timepoints (2, 4, 6, 8, 10, 12 and 14 day) (F 8, 144=41.34, P<0.001; Fig. 1a). The levels of inflammatory factors in the DRG, SC and HIP were altered. Both mRNA and protein levels of TNF-α on the eighth and fourteenth days after surgeries were significantly increased in DRG, SC and HIP of the CCI group compared with those of the sham group (DRG: day 8, P=0.0051, day 14, P=0.00057; SC: day 8, P=0.0090, day 14, P=0.0018; HIP: day 8, P=0.0055, day 14, P=0.0019; Fig. 1b. and DRG: day 8, P=0.016, day 14, P=0.0036; SC: day 8, P=0.012, day 14, P=0.0073; HIP: day 8, P=0.032, day 14, P=0.012; Fig. 1d.). The mRNA and protein levels of IFN-γ showed a trend similar to that of TNF-α (DRG: day 8, P=0.00244, day 14, P=0.00029, SC: day 8, P=0.00049, day 14, P=0.00096, HIP: day 8, P=0.00424, day 14, P=0.00288, Fig. 1c; DRG: day 8, P=0.053, day 14, P=0.0021, SC: day 8, P=0.016, day 14, P=0.0060, HIP: day 8, P=0.023, day 14, P=0.078, Fig. 1e).
Upregulated expression of receptor-interacting protein 1/receptor-interacting protein 3
We analysed the mRNA expression of RIP1 and RIP3 with RT-PCR, and protein levels of RIP1 and RIP3 by western blotting. In the DRG, SC and HIP, the levels of RIP1 mRNA were significantly increased at 14 d after ligation (DRG: P=0.0018, SC: P=0.0010, HIP: P=0.0031; Fig. 2a). RIP3 was significantly upregulated in the CCI group compared with the sham group (DRG: P=0.015, SC: P=0.0019, HIP: P=0.0066; Fig. 2b). Results of western blotting showed that protein levels of RIP1 and RIP3 were also increased by CCI (Fig. 2c–e).
Activation of inflammasome and nuclear factor-κB signalling
RIP1 and RIP3 modulate inflammasome signalling 27 and regulate the target protein DRP1. Analysis of DRP1 expression showed that DRP1 was induced in the DRG (Fig. 3a), SC (Fig. 3b) and HIP (Fig. 3c). NLRP3, a key protein of inflammasome signalling, was also observed in the DRG, SC and HIP. As shown in Fig. 3, the protein levels of NLRP3 in the DRG, SC and HIP were increased 14 days after the operation. Analysis of protein levels of NF-κB p65 by western blotting showed that NF-κB p65 was upregulated in the DRG, SC and HIP (Fig. 3a–c).
Increased PD1 protein
To assess programmed cell death induced by CCI, the protein levels of PD1 in the DRG, SC and HIP were measured. PD1 was increased in all three areas in the CCI group compared with the sham group (Fig. 4a–c). High levels of PD1 protein reflected that CCI promotes the cell death in these three nerve tissues.
Sciatic nerve CCI can cause neuroinflammation and neuropathic pain because of pathological changes in the central and peripheral nerves. In this study, we reproduced the sciatic nerve CCI model and analysed the levels of TNF-α, IFN-γ, RIP1 and RIP3 in the DRG, SC and HIP. Our results suggest that CCI of the sciatic nerve in mice may increase the expression of RIP1 and RIP3, which play a role in integrating neuroinflammatory responses and necrosis in CCI. Inflammasome signalling was activated by RIP1/RIP3/DRP1 in the DRG, SC and HIP, which may induce chronic inflammatory responses and necrosis. Moreover, the high levels of PD1 protein imply that CCI promotes cell death in these brain regions.
The levels of TNF-α and IFN-γ were also significantly increased. TNF-α induces several events, including activation of transcription factor NF-κB and programmed cell death 28. TNF-α is mainly secreted by macrophages 29. Increased TNF-α levels in the DRG, SC and HIP imply that macrophages may infiltrate these regions, in agreement with previous findings 30. TNF-α facilitates excitatory transmission in the acute phase after nerve injury and contributes towards neuropathic pain 6. Our results showed that both mRNA and protein levels of TNF-α were significantly increased at 8 days after ligation in a time-dependent pattern. Changes in TNF-α at the early stage of injury may induce immune cell infiltration, including macrophages and T-cell. IFN-γ was also upregulated at 8 and 14 days after surgeries. Accumulation of IFN-γ can activate spinal microglia and drive neuropathic pain 31.
An inflammatory cascade was also induced by TNF-α and IFN-γ. Both TNF-α and IFN-γ activate RIP1 and RIP3 32–34. The cellular kinase RIP1 is situated at the converging point of several pathways 35. In this study, RIP1 and RIP3 protein levels were markedly increased in the sciatic nerve CCI group compared with the sham group. We hypothesize that increased TNF-α and IFN-γ secretion induced the expression of RIP1 and RIP3, which subsequently upregulated the expression of DRP1 and NLRP3 19, thus promoting inflammasome formation. In CCI, DRP1 and NLRP3 protein levels were increased in the DRG, SC and HIP. NLRP3 inflammasome activation can induce neuropathic pain 36. As a caspase-1-activating complex, the NLRP3 inflammasome promotes interleukin-1β secretion and triggers necrosis 37. We observed NF-κB signalling activation in the CCI group, which may have been induced by RIP1 38. Increased NF-κB activity may promote inflammatory factor release. Thus, CCI-induced RIP1 and RIP3 may promote an inflammatory cascade and cell death in the nervous system.
In conclusion, we show that levels of TNF-α and IFN-γ were increased in a sciatic nerve CCI model at the 8th day after ligation. Continuous accumulation of TNF-α and IFN-γ may activate RIP1 and RIP3 in the DRG, SC and HIP. RIP1 and RIP3 may induce chronic neuroinflammation and necrosis, possibly through the NLRP3 inflammasome and NF-κB signalling pathways. This study showed that RIP1 and RIP3 were highly expressed in DRG, SC and HIP of CCI mice, and may play an important role in neuropathic pain.
This work was supported by a grant from the National Natural Science Foundation of China. The authors would like to thank Bin Son and Xin Zhang for their comments on the manuscript and their critical reviews of the study proposal.
This work was supported in part by the General Program of National Natural Science Foundation of China (813700933 and 8167237). The funders did not participate in the study design; collection, management, analysis and interpretation of data; writing of the report; and the decision to submit the report for publication.
Shaofeng Pu, Shuangyue Li, and Dongping Du conceived and designed the study. Shaofeng Pu drafted the statistical analysis plan. Shaofeng Pu, Yongming Xu, and Yingying Lv drafted the original protocol. Junzhen Wu participated in study coordination. All authors have read and approved the final version of the manuscript.
Conflicts of interest
There are no conflicts of interest.
1. Mor D, Bembrick AL, Austin PJ, Keay KA. Evidence for cellular injury in the midbrain of rats following chronic constriction injury
of the sciatic nerve
. J Chem Neuroanat 2011; 41:158–169.
2. Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988; 33:87–107.
3. Vos BP, Strassman AM, Maciewicz RJ. Behavioral evidence of trigeminal neuropathic pain following chronic constriction injury
to the rat's infraorbital nerve. J Neurosci 1994; 14:2708–2723.
4. Hu P, Bembrick AL, Keay KA, McLaclan EM. Immune cell involvement in dorsal root ganglia and spinal cord after chronic constriction or transection of the rat sciatic nerve
. Brain Behav Immun 2007; 21:599.
5. Okamoto K, Martin DP, Schmelzer JD, Mitsui Y, Low PA. Pro- and anti-inflammatory cytokine gene expression in rat sciatic nerve chronic constriction injury
model of neuropathic pain. Exp Neurol 2001; 169:386–391.
6. Zhang H, Zhang H, Dougherty PM. Dynamic effects of TNF-α on synaptic transmission in mice over time following sciatic nerve chronic constriction injury
. J Neurophysiol 2013; 110:1663–1671.
7. Wang C, Wang C. Anti-nociceptive and anti-inflammatory actions of sulforaphane in chronic constriction injury
-induced neuropathic pain mice. Inflammopharmacology 2017; 25:99–106.
8. Chu LW, Chen JY, Wu PC, Wu BN. Atorvastatin prevents neuroinflammation in chronic constriction injury
rats through nuclear NFκB downregulation in the dorsal root ganglion and spinal cord. ACS Chem Neurosci 2015; 6:889–898.
9. Chen Y, Chen X, Yu J, Xu X, Wei X, Gu X, et al. JAB1 is involved in neuropathic pain by regulating JNK and NF-κB activation after chronic constriction injury
. Neurochem Res 2016; 41:1119–1129.
10. Zhou J, Wang J, Li W, Wang C, Wu L, Zhang J. Paeoniflorin attenuates the neuroinflammatory response in a rat model of chronic constriction injury
. Mol Med Rep 2017; 15:3179–3185.
11. Riffel AP, de Souza JA, Santos Mdo C, Horst A, Scheid T, Kolberg C, et al. Systemic administration of vitamins C and E attenuates nociception induced by chronic constriction injury
of the sciatic nerve
in rats. Brain Res Bull 2016; 121:169–177.
12. Festjens N, Vanden Berghe T, Cornelis S, Vandenabeele P. RIP1, a kinase on the crossroads of a cell’s decision to live or die. Cell Death Differ 2007; 14:400–410.
13. Ofengeim D, Yuan J. Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death. Nat Rev Mol Cell Biol 2013; 14:727–736.
14. Mack C, Sickmann A, Lembo D, Brune W. Inhibition of proinflammatory and innate immune signaling pathways by a cytomegalovirus RIP1-interacting protein. Proc Natl Acad Sci USA 2008; 105:3094–3099.
15. Sun X, Lee J, Navas T, Baldwin DT, Stewart TA, Dixit VM. RIP3, a novel apoptosis-inducing kinase. J Biol Chem 1999; 274:16871–16875.
16. Moriwaki K, Chan FK. RIP3: a molecular switch for necrosis and inflammation. Genes Dev 2013; 27:1640–1649.
17. Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 2009; 137:1112–1123.
18. Wang X, Jiang W, Yan Y, Gong T, Han J, Tian Z, et al. RNA viruses promote activation of the NLRP3 inflammasome through a RIP1-RIP3-DRP1 signaling pathway. Nat Immunol 2014; 15:1126–1133.
19. Zhou K, Shi L, Wang Z, Zhou J, Manaenko A, Reis C, et al. RIP1-RIP3-DRP1 pathway regulates NLRP3 inflammasome activation following subarachnoid hemorrhage. Exp Neurol 2017; 295:116–124.
20. Obata K, Yamanaka H, Dai Y, Mizushima T, Fukuoka T, Tokunaga A, et al. Differential activation of MAPK in injured and uninjured DRG neurons following chronic constriction injury
of the sciatic nerve
in rats. Eur J Neurosci 2004; 20:2881–2895.
21. Gerard E, Spengler RN, Bonoiu AC, Mahajan SD, Davidson BA, Ding H, et al. Chronic constriction injury
-induced nociception is relieved by nanomedicine-mediated decrease of rat hippocampal tumor necrosis factor. Pain 2015; 156:1320–1333.
22. Xiong Q, He W, Wang H, Zhou J, Zhang Y, He J, et al. Effect of the spinal apelin-APJ system on the pathogenesis of chronic constriction injury
-induced neuropathic pain in rats. Mol Med Rep 2017; 16:1223–1231.
23. Liu L, Yin Y, Li F, Malhotra C, Cheng J. Flow cytometry analysis of inflammatory cells isolated from the sciatic nerve
and DRG after chronic constriction injury
in mice. J Neurosci Methods 2017; 284:47–56.
24. Wan L, Luo A, Yu H, Tian Y. Effect of touch-stimulus on the expression of C-fos and TrkA in spinal cord following chronic constriction injury
of the sciatic nerve
in rats. J Huazhong Univ Sci Technolog Med Sci 2005; 25:219–222.
25. Dellarole A, Morton P, Brambilla R, Walters W, Summers S, Bernardes D, et al. Neuropathic pain-induced depressive-like behavior and hippocampal neurogenesis and plasticity are dependent on TNFR1 signaling. Brain Behav Immun 2014; 41:65–81.
26. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994; 53:55–63.
27. Lukens JR, Vogel P, Johnson GR, Kelliher MA, Iwakura Y, Lamkanfi M, Kanneganti TD. RIP1-driven autoinflammation targets IL-1α independently of inflammasomes and RIP3. Nature 2013; 498:224.
28. Antwerp DJV, Martin SJ, Kafri T, Green DR, Verma IM. Suppression of TNF-a-induced apoptosis by NF-KB. Science 1996; 274:787–789.
29. Witsell AL, Schook LB. Tumor necrosis factor alpha is an autocrine growth regulator during macrophage differentiation. Proc Natl Acad Sci USA 1992; 89:4754–4758.
30. Gómez-Nicola D, Valle-Argos B, Suardíaz M, Taylor JS, Nieto-Sampedro M. Role of IL-15 in spinal cord and sciatic nerve
after chronic constriction injury
: regulation of macrophage and T-cell infiltration. J Neurochem 2008; 107:1741–1752.
31. Tsuda M, Masuda T, Kitano J, Shimoyama H, Tozaki-Saitoh H, Inoue K. IFN-gamma receptor signaling mediates spinal microglia activation driving neuropathic pain. Proc Natl Acad Sci USA 2009; 106:8032–8037.
32. Vandenabeele P, Declercq W, Van Herreweghe F, Vanden Berghe T. The role of the kinases RIP1 and RIP3 in TNF-induced necrosis. Sci Signal 2010; 3:4.
33. Thapa RJ, Nogusa S, Chen P, Maki JL, Lerro A, Andrake M, et al. Interferon-induced RIP1/RIP3-mediated necrosis requires PKR and is licensed by FADD and caspases. Proc Natl Acad Sci USA 2013; 110:3109–3118.
34. Ma W, Tummers B, van Esch EM, Goedemans R, Melief CJ, Meyers C, et al. Human papillomavirus downregulates the expression of IFITM1 and RIPK3 to escape from IFNγ- and TNFα-mediated antiproliferative effects and necroptosis. Front Immunol 2016; 7:5.
35. Meylan E, Tschopp J. The RIP kinases: crucial integrators of cellular stress. Trends Biochem Sci 2005; 30:151–159.
36. Min J, Wu C, Gao F, Xiang H, Sun N, Peng P, et al. Activation of NLRP3 inflammasome in peripheral nerve contributes to paclitaxel-induced neuropathic pain. Mol Pain 2017; 13:1744806917719804.
37. Cullen SP, Kearney CJ, Clancy DM, Martin SJ. Diverse activators of the NLRP3 inflammasome promote IL-1β secretion by triggering necrosis. Cell Rep 2015; 11:1535–1548.
38. Cussonhermance N, Khurana S, Lee TH, Fitzgerald KA, Kelliher MA. RIP1 mediates the trif-dependent toll-like receptor 3- and 4-induced NF-κB activation but does not contribute to interferon regulatory factor 3 activation. J Biol Chem 2005; 280:36560–36566.