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Microglial P2Y12 Signaling Contributes to Cisplatin-induced Pain Hypersensitivity via IL-18-mediated Central Sensitization in the Spinal Cord

  • Author Footnotes
    # These authors contributed equally to this work.
    Xue-Tai Chen
    Footnotes
    # These authors contributed equally to this work.
    Affiliations
    Jiangsu Province Key Laboratory of Anesthesiology and Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou Medical University, Xuzhou, Jiangsu 221002, People's Republic of China

    Department of anesthesiology, The Yancheng Clinical College of Xuzhou Medical University; Department of central labotatory, The First people's Hospital of Yancheng, Yancheng, Jiangsu 224006, People's Republic of China
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  • Author Footnotes
    # These authors contributed equally to this work.
    Li-Ping Chen
    Footnotes
    # These authors contributed equally to this work.
    Affiliations
    Department of Pain Medicine, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221002, People's Republic of China
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  • Li-Jun Fan
    Affiliations
    Jiangsu Province Key Laboratory of Anesthesiology and Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou Medical University, Xuzhou, Jiangsu 221002, People's Republic of China
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  • Hou-Ming Kan
    Affiliations
    Jiangsu Province Key Laboratory of Anesthesiology and Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou Medical University, Xuzhou, Jiangsu 221002, People's Republic of China
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  • Zi-Zhu Wang
    Affiliations
    Jiangsu Province Key Laboratory of Anesthesiology and Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou Medical University, Xuzhou, Jiangsu 221002, People's Republic of China
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  • Bin Qian
    Affiliations
    Department of anesthesiology, The Yancheng Clinical College of Xuzhou Medical University; Department of central labotatory, The First people's Hospital of Yancheng, Yancheng, Jiangsu 224006, People's Republic of China
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  • Zhi-Qiang Pan
    Affiliations
    Jiangsu Province Key Laboratory of Anesthesiology and Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou Medical University, Xuzhou, Jiangsu 221002, People's Republic of China
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  • Wen Shen
    Correspondence
    Corresponding authors: Wen Shen, Tel.: +86 13815311266
    Affiliations
    Department of Pain Medicine, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221002, People's Republic of China
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  • Author Footnotes
    # These authors contributed equally to this work.
Open AccessPublished:January 13, 2023DOI:https://doi.org/10.1016/j.jpain.2023.01.005

      Highlights

      • Microglial P2Y12/IL-18 played a critical role in cisplatin-induced pain hypersensitivity.
      • P2Y12/IL-18 enhanced N-methyl-D-aspartate receptor activation during cisplatin-induced pain.

      Abstract

      Administration of cisplatin and other chemotherapy drugs is crucial for treating tumors. However, cisplatin-induced pain hypersensitivity is still a critical clinical issue, and the underlying molecular mechanisms have remained unresolved to date. In this study, we found that repeated cisplatin treatments remarkedly upregulated the P2Y12 expression in the spinal cord. Expression of P2Y12 was predominant in the microglia. Pharmacological inhibition of P2Y12 expression markedly attenuated the cisplatin-induced pain hypersensitivity. Meanwhile, blocking the P2Y12 signal also suppressed cisplatin-induced microglia hyperactivity. Furthermore, the microglia Src family kinase/p38 pathway is required for P2Y12-mediated cisplatin-induced pain hypersensitivity via the proinflammatory cytokine IL-18 production in the spinal cord. Blocking the P2Y12/IL-18 signaling pathway reversed cisplatin-induced pain hypersensitivity, as well as activation of N-methyl-D-aspartate receptor and subsequent Ca2+-dependent signals. Collectively, our data suggest that microglia P2Y12-SFK-p38 signaling contributes to cisplatin-induced pain hypersensitivity via IL-18-mediated central sensitization in the spinal, and P2Y12 could be a potential target for intervention to prevent chemotherapy-induced pain hypersensitivity.

      Perspective

      Our work identified that P2Y12/IL-18 played a critical role in cisplatin-induced pain hypersensitivity. This work suggests that P2Y12/IL-18 signaling may be a useful strategy for the treatment of chemotherapy-induced pain hypersensitivity.

      Keywords

      Introduction

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      Materials and Methods

      Animals

      Adult male Sprague–Dawley rats (body weight: 180 to 200 g) were purchased from the Experimental Animal Center of Xuzhou Medical University, China. The rats were housed in groups of five rats in each cage with ad libitum access to food and water, in a climate conditioned room at a room temperature of 23°C ± 0.5°C and a 12-h light/dark cycle (light from 7:00 am to 7:00 pm). Before the initiation of experiments, the rats were acclimatized in the animal facility for a minimum of 7 days. All the manipulations of rats for the experiments were performed following the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Ethical Issues of the International Association for the Study of Pain. Experimenters who performed behavioral tests, drug administration, and preparation of spinal cord tissues for Western blot analysis and immunohistochemistry were blinded.

      Establishment of cisplatin-induced pain hypersensitivity

      To establish cisplatin-induced pain hypersensitivity, treatment with cisplatin (dose: 1.5 mg/kg) (Sigma-Aldrich, USA) was performed in two rounds of 4-daily intraperitoneal injection with a 4-day break in between
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      . An equal quantity of normal saline was administered to control rats. The intraperitoneal injections were given with a 30.5– gauge needle fitted 500-μL BD syringe.

      Mechanical Allodynia

      Paw withdrawal threshold was measured in response to von Frey filament stimulation (Aesthesio, Danmic Global, San Jose, CA, USA) to determine the presence of mechanical allodynia. Briefly, following 3 consecutive days of acclimatization, the rat was kept on a wire net floor in a plexiglass chamber and allowed to familiarize for 10–15 min before the experiment is initiated. The rat's mid-plantar surface of the hind paw was applied with a series of filaments (0.4, 0.6, 1.4, 2, 4, 6, 8, 10, and 15 g) with sustaining pressure to elicit a paw withdrawal reflex or to bend the filament for 5 s within 5 s. Application of each of the filaments was done five times, followed by the calculation of the 50% threshold (g) employing the formula: maximum bending force value [(maximum bending force value minimum bending force value) ÷ (positive rate of the maximum bending force − positive rate of the minimum bending force)] × (positive rate of the maximum bending force 50%).

      Cold Allodynia

      For determining sensitivity to cold, acetone (∼50 μL) was smeared to the rat hind paw's lateral plantar surface, using a 1 mL syringe, ensuring that the tip of the syringe did not touch the paw. In the single model, the time taken for lifting, shaking, or licking the treated paw by the rat was recorded during a period of 60 s. Measurements were performed every 5 min and the average value of three repeats was used as the result.

      Administration of Drugs

      P2Y12 inhibitor MRS2395 (M5942) was purchased from Sigma (USA). Src family kinase inhibitor PP2 (S7008) and P38 inhibitor SB239063 (S7741) were obtained from Selleck Chemicals (China). PP3 (an inactive analog of PP2, 2794), IL-18-binding protein (IL-18 BP, an IL-18 antagonist, AF119), rat IL-18 neutralizing antibody (IL-18 Nab, AF521) and control antibody (normal goat immunoglobulin G, AB-108-C) were obtained from R&D Systems (USA). MRS2395, PP2, PP3, and SB239063 were first solubilized in dimethyl sulfoxide and diluted in phosphate-buffered saline (keeping the final concentration of dimethyl sulfoxide at 5%). IL-18 BP, IL-18-neutralizing antibody and control immunoglobulin G were solubilized in phosphate-buffered saline. All surgeries were done using anesthesia with sodium pentobarbital (50 mg/kg, intraperitoneal). All reagents were administered 2 h before cisplatin or saline injection, via intrathecal (i.t.) injection, in a volume of 10 μL, into the cerebral spinal fluid, and the employed doses were determined in previous studies and preliminary experiments
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      . Intrathecal injections were made employing a 10-μL microinjection syringe as described previously. The syringe was inserted between the L5–L6 region of the spinal cord, in the intervertebral space of a conscious rat. The accuracy of each injection was ascertained by a reflexive flick of the rat's tail. Treatment time points and reagent doses are given in Fig. 1.
      Figure 1
      Figure 1Repeated cisplatin treatment-induced pain hypersensitivity, as well as expression and distribution of P2Y12 in the spinal cord. (A) Schematic timeline for drug administration, behavioral detection, and tissue extraction. (B) Cisplatin lowered the paw withdrawal threshold in the von Frey test. Data are shown as the mean ± SEM. *P< 0.05, **P< 0.01 versus saline group, n = 8 per group. Two-way repeated-measures ANOVA with the post hoc Bonferroni test. (C) Cisplatin increased the duration of licking in the acetone test. Data are presented as the mean ± SEM. *P< 0.05 and **P< 0.01, versus saline group, n = 8 per group, two-way repeated-measures ANOVA with the post hoc Bonferroni test. (D) Representative bands and analysis of P2Y12 and IBA-1 protein expression. Data were normalized to β-actin. Data are shown as the mean ± SEM. *P< 0.05, **P< 0.01 vs. −1 day, n = 4 per time point. One-way ANOVA with the post hoc Dunnett test. (E) Immunofluorescence images of the P2Y12 and IBA-1 immunoreactivity in the spinal dorsal horn of rats treated with repetitive saline or cisplatin administration. Tissues were harvested on day 15 post administration. Scale bars: 100 μm. (F, G) Immunofluorescence staining of P2Y12 combined with cell-type-specific markers in the spinal dorsal horn: IBA-1 (microglial marker), NeuN (neuronal marker), GFAP (astroglial marker), and OLIG2 (oligodendrocytic marker). P2Y12 immunoreactivity predominantly colocalized with IBA-1 (F), but not with NeuN, GFAP, or OLIG2 (G). Tissues were harvested on day 15 post administration. Scale bars: 50 μm and 20 μm (zoom). ANOVA = analysis of variance; GFAP = glial fibrillary acidic protein; IBA-1 = ionized calcium-binding adapter molecule 1; IF = immunofluorescence; i.p. = intraperitoneal; NeuN = neuronal nuclei; OLIG2 = oligodendrocyte transcription factor 2; PWT = paw withdrawal threshold; SEM = standard error of the mean; WB = western blotting

      Western Blotting

      L4–L6 spinal dorsal horn tissues were harvested and homogenized in ice-cold lysis buffer for radioimmunoprecipitation assay, containing 1% PMSF and a protease/phosphatase inhibitor cocktail (5872, Cell Signaling Technology). Following incubation for 20 min on ice, the lysate was centrifuged for 20 min at 4°C and 12,000 × g, to isolate the proteins. Bicinchoninic acid protein assay kit (Beyotime, Shanghai, China) was used to determine the protein concentrations in the samples. Protein samples (50 μg) were subjected to heat denaturation for 5 min at 100°C, and then separation of proteins was done on sodium dodecyl sulfate-polyacrylamide gels, followed by transfer onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The PVDF membranes with transferred proteins were blocked for 2 h at room temperature using 5% (w/v) nonfat milk in Tris-buffered saline with 1% Tween (TBST). Then, the membranes were incubated overnight with primary antibodies at 4°C (Table 1). Dilution of all the primary antibodies was done in 5% (w/v) nonfat milk in TBST. After overnight incubation, the membranes were washed using TBST and further incubated with corresponding horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. Protein bands were detected by imaging the signal generated using Enhanced chemiluminescence reagent (Thermo Fisher Scientific, Rockford, IL, USA), and Image Quant Ai600 (General Electric Co., Kenilworth, NJ, USA) for visualizing the image. ImageJ software (version 2.0; National Institutes of Health, Bethesda, MD, USA) was used to analyze and quantify the images. Western blotting analysis of each sample was repeated for at least six times to obtain consistent results.
      Table 1Primary antibodies used in the western blotting analysis
      NameMolecular weightCat. no.ManufacturerDilution
      Rabbit anti-P2Y1250 kDaAS-55043AAnaspec Inc.1:1,000
      Rabbit anti-phospho-Src Family (Tyr 416)60 kDa#6943CST1:2,000
      Rabbit anti-pp38 (Thr180/Tyr182)38 kDa#4511CST1:1,000
      Goat anti-IL-1822 kDaAF521R&D Systems1:1,000
      Goat anti-IBA-117 kDaab5076abcam1:1,000
      Rabbit anti-phospho-NMDAR 2B (Tyr 1472)190 kDa#4208CST1:1,000
      Rabbit anti-phospho-ERK1/2 (Thr 202/Tyr 204)42, 44 kDa#9101CST1:1,000
      Rabbit anti-phospho-CaMKIIα (Thr 286)50 kDa#12716CST1:1,000
      Rabbit anti-phospho-CREB (Ser 133)43 kDa#9198CST1:1,000
      HRP-conjugated GAPDH antibody36 kDaHRP-60004Proteintech1:10,000
      HRP-conjugated beta actin antibody43 kDaHRP-60008Proteintech1:10,000
      CST=Cell Signal Technology; GAPDH=glyceraldehyde-3-phosphate dehydrogenase; HRP=horseradish peroxidase; IBA-1=ionized calcium-binding adapter molecule 1; IL-18=interleukin-18

      Fluorescence Immunohistochemistry

      Rats were anesthetized and transcardially perfused with 4% cold paraformaldehyde on day 15 after the first injection of cisplatin, for conducting fluorescence immunohistochemistry. After harvesting the lumbar spinal cords, they were postfixed in 4% paraformaldehyde for 2 h, at 4°C, followed by sequential dehydration in 10%, 20%, and 30% sucrose overnight for 3 days. Transverse sections of spinal cords were made into 30 μm thick slices, using a cryostat and washed with TBST. Blocking of the tissue sections was done using 0.3% Triton X-100 in 5% donkey serum for 1 h at room temperature followed by overnight incubation at 4°C with the primary antibodies, as detailed in Table 2. The sections were subsequently washed thrice with TBST for 15 min and incubated for 1 h at room temperature with Alexa Fluor 488 or 594 conjugated corresponding secondary antibodies (1:1,000; Invitrogen, Carlsbad, CA, USA). Confocal scanning laser microscope (Fluo View FV1000; Olympus Co., Tokyo, Japan) was employed to capture immunofluorescence images.
      Table 2Primary antibodies used in the immunofluorescence analysis
      NameCat. no.ManufacturerDilution
      Rabbit anti-P2Y12AS-55043AAnaspec Inc.1:500
      Rabbit anti-phospho-Src Family (Tyr 416)#6943CST1:500
      Rabbit anti-pp38 (Thr180/Tyr182)#4511CST1:500
      Goat anti-IL-18AF521R&D Systems1:500
      Goat anti-IL-18RAF856R&D Systems1:500
      Rabbit anti-IBA-1019-19741Wako1:500
      Goat anti-IBA-1NB100-1028Novus1:500
      Mouse anti-GFAP#3670CST1:500
      Mouse anti-NeuNMAB377Millipore1:500
      Mouse anti-OLIG2MABN50Millipore1:500
      CST = Cell Signal Technology; GFAP = glial fibrillary acidic protein; IBA-1 = ionized calcium-binding adapter molecule 1; IL-18 = interleukin-18; NeuN = neuronal nuclei

      Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

      TRIzol reagent (Invitrogen) was used to extract total RNA from L4-6 spinal dorsal horn tissues according to the manufacturer's instructions. Subsequently, using a PrimeScript RT Master Mix (RR036A; Takara, Japan) total RNA (1 μg) was reverse transcribed into cDNA. Each reaction was conducted in triplicate using a SYBR Premix Ex Taq kit (RR420A; Takara) in a final volume of 20 μL containing 2 μL of cDNA and 10 μM of gene-specific primers. The RT-PCR was done on a 7300 Plus Real-Time PCR system (Applied Biosystems, USA) under the thermocycling conditions: 95°C for 30 s followed by 40 amplification cycles (5 s at 95°C and 30 s at 60°C). The mRNA expression levels were analyzed by the 2−∆∆Ct method, using glyceraldehyde-3-phosphate dehydrogenase as an endogenous control. The primer sequences (BioTNT, China) used in this experiment are given in Table 3.
      Table 3Primer sequences used in RT-qPCR
      GenePrimer forward (5′-3′)Primer reverse (5′-3′)
      IL-185′ GCT GTG ACC CTA TCT GTG AAG5′ ATT CCA TTT TGT TGT GTC CTG
      GAPDH5′ GTG CCA GCC TCG TCT CAT AG5′ GAA CTT GCC GTG GGT AGA GT
      GAPDH = glyceraldehyde-3-phosphate dehydrogenase; IL-18 = interleukin-18; RT-qPCR = real-time quantitative polymerase chain reaction

      Statistical Analyses

      All results are shown as the mean ± standard error of the mean. Differences between groups for the pain threshold and latency results from the pain behavioral experiments, were assessed using two-way repeated-measures analysis of variance (ANOVA) followed by the post hoc Bonferroni multiple comparisons test. Intergroup differences for the western blotting, RT-qPCR, and immunofluorescence experiments were determined using one-way ANOVA followed by the post hoc Dunnett multiple comparison test. Differences were considered statistically significant at P-values <0.05 (Table 4 and Table 5). No data were excluded from the statistical analyses and also any outliers were not evaluated. GraphPad Prism8.0 software (GraphPad Software Inc., San Diego, CA, USA) was used for all the statistical analyses.
      Table 4Two-way RM ANOVA
      FigurePanelInteractionTimeTreatment
      DFn,DFdFPDFn,DFdFPDFn,DFdFP
      1B4,289.405<0.00014,285.982=0.00131,780.07<0.0001
      1C1,77.351=0.03011,711.55=0.01151,762.4<0.0001
      2B8,564.195=0.00054,2813.75<0.00012,1425.54<0.0001
      2E8,566.496<0.00014,2821.14<0.00012,1434.84<0.0001
      3E8,567.358<0.00014,2842.45<0.00012,1479.58<0.0001
      3H8,5613.78<0.00014,2824.12<0.00012,1490.68<0.0001
      3F8,564.507=0.00034,2811.56<0.00012.1454.80<0.0001
      3I8,566.530<0.00014,2823.48<0.00012,1439.19<0.0001
      6B8,564.582=0.00024,2821.71<0.00012,1443.47<0.0001
      6C8,565.044<0.00014,289.514<0.00012,1429.19<0.0001
      Table 5Ono-way ANOVA
      FigurePanelFactorTreatment
      DFn,DFdFP
      1DP2Y124,2517<0.0001
      1DIBA-14,258.796=0.0001
      2CBehavior2,2132.72<0.0001
      2FBehavior2,2135.98<0.0001
      3GBehavior3,2812.44<0.0001
      3JBehavior3,2815.94<0.0001
      3Ap-SFK4,155.876=0.0047
      3Ap384,1512.02=0.0001
      4Ap-p382,45127.5<0.0001
      4AIBA-12,45170.8<0.0001
      4Ap-SFK2,4574.60<0.0001
      5DIL-18 mRNA3,1231.64<0.0001
      5EIL-18 mRNA3,1229.23<0.0001
      5FIL-18 mRNA3,1243.80<0.0001
      5HIL-183,1251.28<0.0001
      5JIL-183,1275.76<0.0001
      5IIL-183,1244.30<0.0001
      6DBehavior3,2815.5<0.0001
      7Ap-NR2B2,910.89=0.0040
      7Ap-CaMKII2,918.93=0.0006
      7Ap-ERK2,976.23<0.0001
      7Ap-CREB2,921.500.0004
      7Bp-NR2B2,916.170.0009
      7Bp-CaMKII2,926.630.0002
      7Bp-ERK2,914.990.0014
      7Bp-CREB2,938.35<0.0001

      Results

      Repeated cisplatin treatment-induced pain hypersensitivity and P2Y12 upregulation in the spinal microglia

      Rats received treatment with two rounds of cisplatin (1.5 mg/kg) 4-daily i.p. injections with an intermittent 4-day break (Fig. 1A). Multiple injections of cisplatin led to a reduction in the paw withdrawal threshold on days 11 and 15 following the first cisplatin injection (Fig. 1B). Simultaneously, cisplatin led to an extended duration of paw lifting/licking as noticed in the acetone test (Fig. 1C). These results showed that multiple cisplatin injections led to persistent mechanical and cold allodynia.
      Western blotting results demonstrated that the expressions of P2Y12 and IBA-1 (a microglia marker) were increased from days 7 to 15 after the first injection of cisplatin (Fig. 1D). Similarly, the immunofluorescence results revealed that the expressions of P2Y12 and IBA-1 were also increased on day 15 in the cisplatin-treated groups (Fig. 1E).
      To further detect the cellular localization of P2Y12, we performed immunostaining with different cell-type markers in the spinal dorsal horn on day 15 after the first injection of cisplatin and observed that P2Y12 was specifically co-expressed with IBA-1 in the spinal dorsal horn. In addition, the double immunostaining images excluded the co-localization of P2Y12 with GFAP (an astroglial marker), OLIG2 (an oligodendrocytic marker), and NeuN (a neuronal marker; Fig. 1F).

      Spinal blockade of P2Y12 prevented or alleviated cisplatin-induced pain hypersensitivity

      Subsequently, we used pharmacologic approaches to assess the regulatory role of spinal P2Y12 in the development and maintenance of cisplatin-induced pain hypersensitivity. Previous reports have shown that systematic administration of P2Y12 inhibitor MRS2395 attenuates pain hypersensitivity in some rat models of neuropathy
      • Jing F
      • Zhang Y
      • Long T
      • He W
      • Qin G
      • Zhang D
      • Chen L
      • Zhou J.
      P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine.
      ,
      • Yu T
      • Zhang X
      • Shi H
      • Tian J
      • Sun L
      • Hu X
      • Cui W
      • Du D.
      P2Y12 regulates microglia activation and excitatory synaptic transmission in spinal lamina II neurons during neuropathic pain in rodents.
      . In this study, we further investigated the analgesic effects of MRS2395 on cisplatin-induced pain hypersensitivity. According to the experimental design and timeline (Fig. 2A), repeated pre-administration of MRS2395 (2 μg, intrathecally injected once daily on days 0, 1, 2, 3, 8, 9, 10, and 11) significantly attenuated the mechanical allodynia on days 3, 11, and 15 (Fig. 2B) and cold allodynia on day 15 (Fig. 2C). Furthermore, according to the experimental design and timeline (Fig. 2D), repeated administration of MRS2395 (2 μg, intrathecal injection once daily from days 11 to 15) reversed the mechanical and cold allodynia on day 15 (Fig. 2E, F, respectively) after the first injection of cisplatin at the late phase. Thus, these behavioral results demonstrated that MRS2395 partially prevented or alleviated the cisplatin-induced mechanical and cold allodynia.
      Figure 2
      Figure 2Spinal blockade of P2Y12 attenuates mechanical and cold allodynia in cisplatin-treated rats. (A) Schematic timeline for drug administration and behavioral detection. Pharmacological inhibition of P2Y12 with MRS2395 (2 μg, intrathecally) or vehicle control dimethyl sulfoxide (DMSO) (vehicle control for MRS2395, 5%, intrathecally) was injected daily once, on days 0, 1, 2, 3, 8, 9, 10, and 11 after the first cisplatin injection. Behavioral tests were conducted 4 h following each injection. (B, C) Intrathecal injection of MRS2395 significantly prevented cisplatin-induced mechanical allodynia (B) and cold allodynia (C). Data are shown as the mean ± SEM. *P< 0.05 and **P< 0.01, compared with the cisplatin + DMSO group; n = 8 per group, two-way repeated-measures ANOVA with the post hoc Bonferroni test.(D) Schematic timeline for drug administration and behavioral detection. MRS2395 (2 μg, intrathecally) or DMSO (5%, intrathecally) was injected once daily on days 12, 13, 14, and 15 after the first cisplatin injection. Behavioral tests were conducted 4 h after each injection. (E, F) Intrathecal injection of MRS2395 markedly attenuated cisplatin-induced mechanical allodynia (E) and cold allodynia (F). Data are presented as the mean ± SEM. *P< 0.05 and **P< 0.01, compared with the cisplatin + DMSO group, n = 8 per group, two-way repeated-measures ANOVA with the post hoc Bonferroni test. ANOVA = analysis of variance; DMSO = dimethyl sulfoxide; i.p. = intraperitoneal; PWT = paw withdrawal threshold; SEM = standard error of the mean

      Microglial src family kinase (SFK) and p38 pathways are functionally activated and involved in P2Y12-mediated cisplatin-induced pain hypersensitivity

      We subsequently examined the downstream molecular signaling mechanisms of P2Y12 in cisplatin treatment rats. Earlier studies have shown that the activity of the SFK pathway can amplify phospho-p38 (p-p38) pathways in spinal microglia in different chronic pain models
      • Chen G
      • Zhang YQ
      • Qadri YJ
      • Serhan CN
      • Ji RR.
      Microglia in Pain: Detrimental and Protective Roles in Pathogenesis and Resolution of Pain.
      ,
      • Ji RR
      • Strichartz G.
      Cell signaling and the genesis of neuropathic pain.
      . In this study, we found that the p-SFK and p-p38 pathways exhibited the same time pattern with the expression of P2Y12 after treatment with cisplatin (Fig. 3A, B). Western blotting results demonstrated that p-SFK and p-p38 expression was increased from day 7 to 15 in the cisplatin-treated group (Fig. 3A). Similarly, the immunofluorescence results showed that p-SFK and p-p38 were expressed in reduced levels in the spinal dorsal horn of saline-treated rats; however, their expression levels were markedly elevated in the spinal dorsal horn of cisplatin-treated rats (Fig. 3B). Double immunostaining indicated that the expression of p-SFK and p-p38 was high in the dorsal horn after treatment with cisplatin and exclusively colocalized with IBA-1 (Fig. 3C, D), but not with NeuN or GFAP.
      Figure 3
      Figure 3Microglial Src family kinase (SFK) and p38 pathways are functionally expressed in the microglia after repeated cisplatin treatments. (A) Cisplatin-stimulated the increase in phospho-SFK (p-SFK) and p-p38 in a time-dependent manner. Data are shown as the mean ± SEM. *P< 0.05, **P< 0.01 versus -1 day. n = 4 per time point, one-way ANOVA with the post hoc Dunnett test. (B) Immunofluorescence images of the p-SFK and p-p38 immunoreactivity in the spinal dorsal horn of rats treated with repetitive saline or cisplatin administration. Tissues were harvested on day 15 post administration. Scale bars: 100 μm. (C, D) Immunoreactivity showed that p-SFK and p-p38 were colocalized with IBA-1, but not with NeuN or GFAP in the spinal dorsal horn. Scale bars: 50 μm and 20 μm (zoom). (E–G) PP2 (10 μg, intrathecally), PP3 (an inactive analog of PP2, 10 μg, intrathecally), SB239063 (10 μg, intrathecally), or DMSO (5%, intrathecally) was injected daily once, on days 0, 1, 2, 3, 8, 9, 10, and 11 after the first cisplatin injection. Behavioral tests were conducted 4 h following each injection. Repeated pre-administration of SFK inhibitor PP2 (E) or p38 inhibitor SB239063 (F) prevented cisplatin-induced mechanical allodynia. Data are presented as the mean ± SEM. *P< 0.05 and **P< 0.01, in comparison with cisplatin + PP3 (or DMSO) group, n = 8 per group, two-way repeated-measures ANOVA with the post hoc Bonferroni test. (G) PP2 and SB239063 tended to relieve cold allodynia, although the difference was not statistically significant. Data are shown as the mean ± SEM. n = 8 per group, one-way ANOVA with the post hoc Dunnett test. (H–J) PP2 (10 μg, intrathecally), PP3 (10 μg, intrathecally), SB239063 (10 μg, intrathecally), or DMSO (5%, intrathecally) was injected daily once, on days 12, 13, 14, and 15 after the first cisplatin injection. Behavioral tests were conducted 4 h following each injection. The repeated administration of PP2 (H) or SB239063 (I) alleviated cisplatin-induced mechanical allodynia. Data are shown as the mean ± SEM. *P< 0.05 and **P< 0.01, in comparison with the cisplatin + PP3 (or DMSO) group, n = 8 per group, two-way repeated-measures ANOVA with the post hoc Bonferroni test. (J) PP2 or SB239063 alleviated cold allodynia. Data are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01, compared with the cisplatin group, n = 8 per group, one-way ANOVA with the post hoc Dunnett test. ANOVA = analysis of variance; DMSO = dimethyl sulfoxide; GFAP = glial fibrillary acidic protein; IBA-1 = ionized calcium-binding adapter molecule 1; NeuN = neuronal nuclei; SEM = standard error of the mean; PWT = paw withdrawal threshold; SFK = Src family kinase
      Furthermore, repeated pre-administration of SFK inhibitor PP2 or p38 inhibitor SB239063 prevented cisplatin-induced mechanical allodynia (Fig. 3E, F). PP2 or SB239063 tended to relieve cold allodynia on day 15 after treatment with cisplatin, although the difference was not statistically significant (Fig. 3G). Repeated administration of PP2 or SB239063 alleviated mechanical and cold allodynia on day 15 after the first injection of cisplatin (Fig. 3H-J).
      Subsequently, the immunofluorescence results showed that intrathecal administration of MRS2395 significantly decreased the cisplatin-increased fluorescence intensity of IBA-1, as well as p-SFK, and number of p-p38-positive cells in the spinal cord (Fig. 4A, B). These findings suggest that the SFK/p38 pathway is a functional downstream factor of P2Y12-mediated cisplatin-induced pain hypersensitivity.
      Figure 4
      Figure 4P2Y12 is required for the Src family kinase (SFK)/p38 and microglial activation after repeated cisplatin treatments. (A, B) Spinal injection of P2Y12 inhibitor MRS2395 significantly suppressed the cisplatin-induced increase in mean fluorescence intensity of IBA-1 and phospho-SFK (p-SFK) and number of p-p38-positive cells. Scale bars: 100 μm. MRS2395 (2 μg, intrathecally) or DMSO (5%, intrathecally) was administered by injections daily once, on days 12, 13, 14, and 15 after the first cisplatin injection. Tissues were collected 4 h after the last injection on day 15. Data are shown as the mean ± SEM. **P < 0.01, in comparison with the cisplatin + DMSO group, n = 16 slices from four rats per group, one-way ANOVA with the post hoc Dunnett test. ANOVA = analysis of variance; DMSO = dimethyl sulfoxide; IBA-1 = ionized calcium-binding adapter molecule 1; SEM = standard error of the mean; SFK = Src family kinase

      P2Y12 signaling pathways promote cisplatin-induced IL-18 production in the spinal microglia

      Earlier studies have shown that activation of microglia stimulates robust production of proinflammatory cytokines, like IL-18, that are implicated in the pathogenesis of neuropathic pain
      • Hu XM
      • Yang W
      • Du LX
      • Cui WQ
      • Mi WL
      • Mao-Ying QL
      • Chu YX
      • Wang YQ.
      Vascular Endothelial Growth Factor A Signaling Promotes Spinal Central Sensitization and Pain-related Behaviors in Female Rats with Bone Cancer.
      ,
      • Liu S
      • Liu YP
      • Lv Y
      • Yao JL
      • Yue DM
      • Zhang MY
      • Qi DY
      • Liu GJ.
      IL-18 Contributes to Bone Cancer Pain by Regulating Glia Cells and Neuron Interaction.
      ,
      • Yang Y
      • Li H
      • Li TT
      • Luo H
      • Gu XY
      • Lü N
      • Ji RR
      • Zhang YQ.
      Delayed activation of spinal microglia contributes to the maintenance of bone cancer pain in female Wistar rats via P2X7 receptor and IL-18.
      . In agreement with the earlier studies, immunoreactivity of IL-18 was primarily colocalized with IBA-1 (Fig. 5A), but not with neurons (NeuN) or astrocytes (GFAP; Fig. 5B). In addition, we further confirmed that IL-18 was obviously co-expressed with P2Y12, p-SFK, and p-p38 (Fig. 5C). Thus, we hypothesized that P2Y12 signaling facilitates the production of proinflammatory cytokine IL-18 via the SFK and p38 pathways in the spinal microglia. To investigate this microglial mechanism of P2Y12/SFK /p38 in cisplatin-induced pain hypersensitivity, we further assessed the changes in Il-18 mRNA expression level in the spinal dorsal horn via spinal blockade of P2Y12, SFK, or p38. RT-qPCR analysis revealed that the mRNA expression levels of Il-18 were pronouncedly increased on day 15. However, the intrathecal administration of P2Y12 inhibitor MRS2395 (Fig. 5D), SFK inhibitor PP2 (Fig. 5E), or p38 inhibitor SB239063 (Fig. 5F) effectively reduced the cisplatin-induced mRNA expression of Il-18. Similarly, the western blotting analysis also showed that MRS2395 (Fig. 5G), PP2 (Fig. 5H), or SB239063 (Fig. 5I) significantly attenuated the cisplatin-induced increase in the expression of IL-18 in the spinal dorsal horn. These findings indicate that the IL-18 expression level was increased after repeated cisplatin treatment and was regulated by the P2Y12/SFK/p38 signaling pathways.
      Figure 5
      Figure 5Spinal blockade of the P2Y12/Src family kinase (SFK)/p38 pathway suppresses the cisplatin-induced IL-18 production. (A) IL-18 immunoreactivity was colocalized with IBA-1, (B) but not with NeuN or GFAP in the spinal dorsal horn. (C) IL-18 immunoreactivity was colocalized with P2Y12, p-SFK, and p-p38 in the spinal dorsal horn. Tissues were harvested on day 15 post administration. Scale bars: 50 μm and 20 μm (zoom). (D–F) Real-time qPCR analyses showed that MRS2395 (D), PP2 (E) or SB239063 (F) significantly attenuated the cisplatin-induced upregulation of IL-18 mRNA expression in the spinal dorsal horn. (G–I) Western blotting analyses showed that MRS2395 (G), PP2 (H) or SB239063 (I) significantly attenuated the cisplatin-induced upregulation of IL-18 protein expression in the spinal dorsal horn. MRS2395 (2 μg, intrathecally), PP2 (10 μg, intrathecally), PP3 (10 μg, intrathecally) or SB239063 (10 μg, intrathecally) was injected daily once, on days 12, 13, 14, and 15 after the first cisplatin injection. Tissues were harvested 4 h following the last injection on day 15. Data are shown as the mean ± SEM. **P < 0.01, in comparison with the saline + DMSO (or PP3) group; ##P < 0.01, compared with the cisplatin + DMSO (or PP3) group, n = 4 per group, one-way ANOVA with the post hoc Dunnett test. ANOVA = analysis of variance; DMSO = dimethyl sulfoxide; GFAP = glial fibrillary acidic protein; IBA-1 = ionized calcium-binding adapter molecule 1; IL-18 = interleukin-18; NeuN = neuronal nuclei; qPCR = quantitative polymerase chain reaction; SEM = standard error of the mean; SFK = Src family kinase

      Spinal blockade of the IL-18/IL-18R axis reduced cisplatin-induced pain hypersensitivity

      Studies have shown that proinflammatory cytokine IL-18 expression has been found to promote the pathogenesis of pain hypersensitivity by binding to its receptor IL-18R
      • Yang Y
      • Li H
      • Li TT
      • Luo H
      • Gu XY
      • Lü N
      • Ji RR
      • Zhang YQ.
      Delayed activation of spinal microglia contributes to the maintenance of bone cancer pain in female Wistar rats via P2X7 receptor and IL-18.
      . In agreement with the earlier studies, immunoreactivity of IL-18R was primarily colocalized with neurons (NeuN) and astrocytes (GFAP), but not with IBA-1 (Fig. 6A). In addition, we assessed the regulatory role of IL-18 in the maintenance of cisplatin-triggered pain hypersensitivity. The repeated spinal injection of IL-18 inhibitor IL-18 BP (1 μg, intrathecally) once daily from days 11 to 15 after treatment with cisplatin significantly reversed the mechanical allodynia on day 15 after the first injection of cisplatin (Fig. 6B). Similarly, IL-18 NAb (1 μg, intrathecally) once daily from days 11 to 15 after treatment with cisplatin also significantly attenuated the mechanical allodynia on day 15 after the first injection of cisplatin (Fig. 6C). Besides, both IL-18 BP and IL-18 NAb significantly reversed the cold allodynia on day 15 after the first injection of cisplatin (Fig. 6D). Thus, spinal blockade of IL-18 partially reduced the cisplatin-induced mechanical and cold allodynia. These results indicate that the microglial mechanism of P2Y12 in the regulation of cisplatin-induced pain hypersensitivity is dependent on the SFK/p38-mediated production of proinflammatory IL-18.
      Figure 6
      Figure 6Spinal blockade of the IL-18/IL-18R axis reduces cisplatin-induced pain hypersensitivity. (A) Double immunostaining of IL-18R and NeuN, GFAP, or IBA-1 in the spinal dorsal horn after repeated cisplatin treatments. Tissues were harvested on day 15 post administration. Scale bars: 50 μm and 20 μm (zoom). (B, C) The repeated administration of IL-18 BP (B) or IL-18 NAb (C) alleviated cisplatin-induced mechanical allodynia. IL-18 BP (1 μg, intrathecally), IL-18 NAb (1 μg, intrathecally), or IgG (1 μg, intrathecally) was injected daily once on days 12, 13, 14, and 15 after the first cisplatin injection. Behavioral tests were conducted 4 h following each injection. Data are shown as the mean ± SEM. *P < 0.05 and **P < 0.01, in comparison with the cisplatin + PBS (or IgG) group, n = 8 per group, two-way repeated-measures ANOVA with the post hoc Bonferroni test. (D) The repeated administration of IL-18 BP or IL-18 NAb alleviated cisplatin-induced cold allodynia. IL-18 BP (1 μg, intrathecally) and IL-18 NAb (1 μg, intrathecally) were injected daily once, on days 12, 13, 14, and 15 after the first cisplatin injection. Behavioral tests were conducted 4 h following each injection. Data are presented as mean ± SEM. *P < 0.05 and **P < 0.01, in comparison with the cisplatin group, n = 8 per group, one-way ANOVA with the post hoc Dunnett test. ANOVA = analysis of variance; BP = binding protein; IgG = immunoglobulin G; IL-18 = interleukin-18; IL-18R = IL-18 receptor; NAb = neutralizing monoclonal antibodies; PBS = phosphate-buffered saline; PWT = paw withdrawal threshold; SEM = standard error of the mean

      P2Y12/IL-18 enhanced N-methyl-D-aspartate receptor activation in the spinal neurons

      Earlier studies have revealed that IL-18 pathway can facilitate neuronal plasticity via interactions with neuronal glutamate receptor and play an essential role in the onset of bone cancer pain
      • Liu S
      • Liu YP
      • Lv Y
      • Yao JL
      • Yue DM
      • Zhang MY
      • Qi DY
      • Liu GJ.
      IL-18 Contributes to Bone Cancer Pain by Regulating Glia Cells and Neuron Interaction.
      . During the formation of chronic pain, N-methyl-D-aspartate (NMDA) receptors in the postsynaptic neuronal membranes are expressed extensively at the spinal cord level. Following their stimulation, NMDA receptors can elevate the intracellular secondary messenger Ca2+ levels and enhance the downstream Ca2+-dependent signaling molecules (e.g., calcium/calmodulin-dependent protein kinase II [CaMKII], extracellular signal-regulated kinase [ERK], and cyclic-AMP response element-binding protein [CREB]), which have an important role in producing and maintaining central sensitization. Among them, ERK and CaMKII elevate neuronal excitability through the phosphorylation of AMPA and NMDA receptors. Besides, CREB, which is a transcription factor, enhances the synthesis of NMDA receptor ligands. Therefore, we hypothesized that P2Y12/IL-18 signaling enables the stimulation of NMDA receptors and Ca2+-triggered signals in spinal neurons. To investigate this neuronal mechanism of P2Y12/IL-18 in cisplatin-induced pain hypersensitivity, we further examined the changes of p-NMDA receptor subunit 2B (p-NR2B; Tyr1472), p-ERK (Thr202/Tyr204), p-CaMKII (Thr286), and p-CREB (Ser133) expression levels in the spinal dorsal horn via spinal blockade of P2Y12 or IL-18. As revealed by western blotting analysis, the expression of p-NR2B, p-ERK, p-CaMKII, and p-CREB proteins were all pronouncedly increased on day 15 after the first injection of cisplatin. However, the intrathecal administration of P2Y12 inhibitor MRS2395 or IL-18 BP effectively reduced the cisplatin-induced protein expression of p-NR2B, p-ERK, p-CaMKII, and p-CREB (Fig. 7A, B). Collectively, these observations suggest that P2Y12/IL-18 signaling likely contributes to cisplatin-induced pain hypersensitivity by controlling neuronal activity via the NMDA receptor.
      Figure 7
      Figure 7Spinal blockade of P2Y12/IL-18 suppresses the cisplatin-induced activation of the NMDA receptor. (A, B) Western blotting analysis showed that MRS2395 (A) or IL-18 BP (B) attenuated the cisplatin-induced upregulation of the expression of phospho-NMDA receptor subunit 2B (p-NR2B) protein, p-extracellular signal-regulated kinase 1/2 (ERK1/2), p-calcium/calmodulin-dependent protein kinase II (CaMKII), and p-cyclic-AMP response element-binding protein (CREB) in the spinal dorsal horn. MRS2395 (2 μg, intrathecally) or IL-18 BP (1 μg, intrathecally) was injected daily once on days 12, 13, 14, and 15 after the first cisplatin injection. Tissues were harvested 4 h after the last injection on day 15. Data are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01, compared with the saline + DMSO (or PBS) group; #P < 0.05 and ##P < 0.01, compared with the cisplatin + DMSO (or PBS) group, n = 4 per group, one-way ANOVA with the post hoc Bonferroni test. ANOVA = analysis of variance; BP = binding protein; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; IL-18 = interleukin-18; NMDA = N-methyl-D-aspartate; PBS = phosphate-buffered saline; SEM = standard error of the mean

      Discussion

      CIPN is a major neuropathological disorder caused by first-line chemotherapy agents
      • Hershman DL
      • Lacchetti C
      • Dworkin RH
      • Lavoie Smith EM
      • Bleeker J
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      • Schneider B
      • Smith ML
      • Smith T
      • Terstriep S
      • Wagner-Johnston N
      • Bak K
      • Loprinzi CL
      Prevention and management of chemotherapy-induced peripheral neuropathy in survivors of adult cancers: American Society of Clinical Oncology clinical practice guideline.
      . Cisplatin and other chemotherapy drugs are widely used in tumor management. However, with prolonged use, cisplatin can cause an intractable hypersensitivity called cisplatin-induced hyperalgesia and decreased anti-tumor efficacy, which limit their clinical usage
      • Banach M
      • Juranek JK
      • Zygulska AL.
      Chemotherapy-induced neuropathies-a growing problem for patients and health care providers.
      ,
      • Seretny M
      • Currie GL
      • Sena ES
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      • Grant R
      • MacLeod MR
      • Colvin LA
      • Fallon M.
      Incidence, prevalence, and predictors of chemotherapy-induced peripheral neuropathy: A systematic review and meta-analysis.
      .
      The present study shows an important role of P2Y12 signaling in cisplatin-induced pain hypersensitivity and supports the view that cisplatin-induced stimulation of P2Y12 signaling may lead to cisplatin-induced pain hypersensitivity by facilitating glial-neuronal interactions. The main observations are as follows: (1) Cisplatin treatment led to a rapid-onset and long-lasting enhancement of P2Y12 in the spinal cord, and subsequent spinal blockade of P2Y12 suppressed microglial activation and prevented or alleviated cisplatin-induced pain hypersensitivity. (2) The p-SFK or p-p38 expression time-dependently increased after cisplatin treatments, which was reduced by the P2Y12 inhibitor MRS2395. In addition, i.t. injection of the SFK inhibitor PP2 or p38 inhibitor SB239063 could significantly prevent or alleviate cisplatin-induced pain hypersensitivity. (3) Cisplatin also led to a marked increase in IL-18 expression in spinal microglial cells. Meanwhile, blocking P2Y12, SFK, or p38 significantly lowered the IL-18 mRNA and protein expressions in cisplatin-treated rats. (4) Inhibiting the P2Y12/IL-18 signaling pathway suppressed the activation of NMDA receptors and subsequent Ca2+-dependent signaling induced by cisplatin, in addition to inhibiting cisplatin-related pain behaviors. These observations suggest a novel mechanism underlying cisplatin-induced pain hypersensitivity and a potential novel therapeutic target for cisplatin-induced pain hypersensitivity.
      In recent years, the role of central glia in CIPN has been discussed in numerous publications
      • Cavaletti G
      • Marmiroli P.
      Chemotherapy-induced peripheral neurotoxicity.
      ,
      • Starobova H
      • Vetter I.
      Pathophysiology of Chemotherapy-Induced Peripheral Neuropathy.
      ,
      • Zajaczkowska R
      • Kocot-Kepska M
      • Leppert W
      • Wrzosek A
      • Mika J
      • Wordliczek J.
      Mechanisms of Chemotherapy-Induced Peripheral Neuropathy.
      . Previous data showed that treatment of rodents with multiple doses of cisplatin-induced pain hypersensitivity, which is close to what is reported clinically
      • Authier N
      • Balayssac D
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      The response of spinal microglia to chemotherapy-evoked painful peripheral neuropathies is distinct from that evoked by traumatic nerve injuries.
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      The mechanisms of microgliosis and pain following peripheral nerve injury.
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      Glia and pain: is chronic pain a gliopathy?.
      .
      Previous animal research on neuropathic, inflammatory, and cancer pain has revealed that the density of spinal microglial P2Y12 is significantly elevated
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      Neurons and glial cells differentially express P2Y receptor mRNAs in the rat dorsal root ganglion and spinal cord.
      ,
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      P2Y12 receptor upregulation in activated microglia is a gateway of p38 signaling and neuropathic pain.
      ,
      • Yu T
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      . Our findings revealed that the P2Y12 expression time-dependently increased in the spinal dorsal horn, correlating with cisplatin-induced mechanical and cold allodynia during repeated cisplatin treatments. P2Y12 expression was mainly confined to the microglia. Cisplatin-induced pain hypersensitivity was greatly attenuated in rats receiving i.t. injection of the P2Y12 inhibitor MRS2395. Meanwhile, the cisplatin-induced activation of microglia was evidently lowered by repeated intrathecal injections of MRS2395. These findings suggest an essential role of P2Y12 signaling in spinal microglial cells in driving cisplatin-induced pain hypersensitivity. Our findings suggest that i.t. injection of the P2Y12 inhibitor MRS2395 could be potentially used to prevent and/or treat the side effects of repeated chemotherapy treatments.
      Gender differences in pain have been extensively studied, and the results of various studies have shown that male and female not only perceive pain differently, but also respond differently to analgesic drugs
      • Greenspan JD
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      • Berkley KJ
      • Fillingim RB
      • Gold MS
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      • Mogil JS
      • Murphy AZ
      • Traub RJ
      Consensus Working Group of the Sex G, Pain SIGotI. Studying sex and gender differences in pain and analgesia: a consensus report.
      . In different animal models of pain, female tend to be more sensitive to pain
      • Mogil JS.
      Qualitative sex differences in pain processing: emerging evidence of a biased literature.
      . Studies have shown that differences in immune systems between different genders may be related to their different responses to pain. Male mice mediate pain through microglia of the spinal cord, while female mice preferentially mediate pain through immune cells, like T-lymphocytes
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      . Considering that our research focused on the role of microglial P2Y12 in cisplatin-induced chemotherapy pain, we conducted experiments using male rats to rule out the effect of sex on the results.
      Previous reports on various chronic pain models demonstrated that SFK is mainly expressed in microglial cells and participates in the activation of the p38 signaling pathways, which are the main mechanisms at the molecular level involved in inflammatory responses mediated by microglia
      • Ji RR
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      Cell signaling and the genesis of neuropathic pain.
      ,
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      p38 MAPK, microglial signaling, and neuropathic pain.
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      Activation of p38 MAP kinase in the rat dorsal root ganglia and spinal cord following peripheral inflammation and nerve injury.
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      ,
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      ,
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      Activation of p38 mitogen-activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury.
      . Our results confirmed that p-SFK or p-p38 was exclusively expressed in the spinal microglia and time-dependently increased after cisplatin treatments, which was reduced by the P2Y12 inhibitor MRS2395. I.t. injection of the SFK inhibitor PP2 or p38 inhibitor SB239063 could significantly prevent or alleviate cisplatin-induced pain hypersensitivity. Thus, SFK/p38 in the spinal cord is a key downstream P2Y12 signaling in the involvement of cisplatin-induced pain hypersensitivity.
      Our studies suggest that P2Y12 signaling operates in a higher hierarchy in driving proinflammatory cytokine cascades that induce cisplatin-induced pain hypersensitivity. The proinflammatory cytokine IL-18 (also known as interferon-γ-inducing factor) has been found to be associated with the pathogenesis of neuropathic pain
      • Hu XM
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      Vascular Endothelial Growth Factor A Signaling Promotes Spinal Central Sensitization and Pain-related Behaviors in Female Rats with Bone Cancer.
      ,
      • Kawasaki Y
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      Cytokine mechanisms of central sensitization: distinct and overlapping role of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in regulating synaptic and neuronal activity in the superficial spinal cord.
      ,
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      Delayed activation of spinal microglia contributes to the maintenance of bone cancer pain in female Wistar rats via P2X7 receptor and IL-18.
      . Specifically, IL-18 is constitutively expressed in microglia in the central nervous system and directly enhances the excitability of neurons by binding to its specific receptor IL-18R
      • Alboni S
      • Cervia D
      • Ross B
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      • Marcondes MC
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      Mapping of the full length and the truncated interleukin-18 receptor alpha in the mouse brain.
      ,
      • Liu S
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      IL-18 Contributes to Bone Cancer Pain by Regulating Glia Cells and Neuron Interaction.
      ,
      • Yang Y
      • Li H
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      Delayed activation of spinal microglia contributes to the maintenance of bone cancer pain in female Wistar rats via P2X7 receptor and IL-18.
      .Consistent with previous studies, IL-18 was exclusively expressed in the spinal microglia, co-localized primarily with P2Y12, p-SFK, and p-p38. In addition, IL-18R was mainly distributed in the spinal neurons and astrocytes. The data yielded from the RT-qPCR and western blotting analyses showed that spinal blockade of P2Y12, SFK, or p38 could markedly suppress the cisplatin-induced mRNA and protein expression of IL-18 in cisplatin-treated rats. I.t. injection of IL-18 BP or IL-18 neutralizing monoclonal antibodies could significantly alleviate cisplatin-induced pain hypersensitivity. These findings suggest that microglial P2Y12 signaling mediates IL-18 production via activation of the SFK/p38 pathway in the spinal cord.
      At signaling levels (Fig. 8), we found that P2Y12 signaling facilitates the production of proinflammatory cytokine IL-18 via the SFK and p38 pathways in the spinal microglia. IL-18 further induced the activation of the NMDA receptors and Ca2+-dependent signals on neurons. Stimulation of NMDA receptor in spinal cord is mainly involved in the generation and maintenance of persistent pain
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      ,
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      . Recent reports suggest that IL-18 released from microglia may enhance NMDA receptor phosphorylation and that intracellular calcium release mediated by NMDA receptor occurs in sensory neurons
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      ,
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      Delayed activation of spinal microglia contributes to the maintenance of bone cancer pain in female Wistar rats via P2X7 receptor and IL-18.
      . Therefore, we further focused on the effect of the P2Y12/IL-18 signaling pathway on the stimulation of NMDA receptors, and the downstream Ca2+-dependent signals in the spinal cord following cisplatin treatment. Western blotting analysis revealed that spinal blockade of P2Y12 or IL-18 markedly suppressed the cisplatin-induced protein expression of p-NR2B, p-CaMKII, p-ERK1/2 and p-CREB in cisplatin-treated rats. Furthermore, recent reports have shown that spinal administration of IL-18 significantly not only elicited mechanically evoked responses of spinal WDR neurons in vivo but also increased the frequency of mEPSCs in spinal IIo neurons
      • Liu S
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      IL-18 Contributes to Bone Cancer Pain by Regulating Glia Cells and Neuron Interaction.
      . Together, these results above support our hypothesis that IL-18 was released from spinal microglia, acts on IL-18R on the neurons by the “ligand-receptor” formation, and mediates central sensitization in the spinal by microglia-to-neurons cross-talk during the initiation and maintenance of cisplatin-induced pain hypersensitivity. However, IL-18R is also expressed in astrocytes in the spinal dorsal horn, whether IL-18R is also responsible for astrocyte activation contributing to cisplatin-induced pain hypersensitivity requires further study.
      Figure 8
      Figure 8Schematic illustration of microglia-neuron interactions in the superficial spinal dorsal horn of rats with cisplatin-induced pain. P2Y12 signaling facilitates the production of proinflammatory cytokine IL-18 via the SFK and p38 pathways in the spinal microglia, which leads to stimulated excitatory synaptic transmission and neuronal hyperactivity in the dorsal horn. These neuromodulation processes in the spinal pain circuit enhance pain sensitivity. ATP = adenosine triphosphate; Glu = glutamate; IL-18 = interleukin-18; IL-18R = IL-18 receptor; NMDAR = N-methyl-D-aspartate receptor; SFK = Src family kinase
      Minocycline is a potent inhibitor of microglial activation, which belongs to a broad-spectrum semi-synthetic bacteriostatic tetracycline, receiving a lot of attention recently in the pain field
      • He L
      • Xu R
      • Chen Y
      • Liu X
      • Pan Y
      • Cao S
      • Xu T
      • Tian H
      • Zeng J.
      Intra-CA1 Administration of Minocycline Alters the Expression of Inflammation-Related Genes in Hippocampus of CCI Rats.
      ,
      • Suzuki H
      • Sugimura Y
      • Iwama S
      • Suzuki H
      • Nobuaki O
      • Nagasaki H
      • Arima H
      • Sawada M
      • Oiso Y.
      Minocycline prevents osmotic demyelination syndrome by inhibiting the activation of microglia.
      . The mechanisms of minocycline analgesia are complex and may involve the inhibition of microglia, the reduction of inflammatory cells, the decrease of neurons excitability and the inhibition of MAPKs signaling pathway
      • Kobayashi K
      • Imagama S
      • Ohgomori T
      • Hirano K
      • Uchimura K
      • Sakamoto K
      • Hirakawa A
      • Takeuchi H
      • Suzumura A
      • Ishiguro N
      • Kadomatsu K.
      Minocycline selectively inhibits M1 polarization of microglia.
      . Recent research indicated a potential usefulness of minocycline used alone or combination with duloxetine in the treatment of oxaliplatin-induced pain
      • Salat K
      • Furgala-Wojas A
      • Salat R.
      The Microglial Activation Inhibitor Minocycline, Used Alone and in Combination with Duloxetine, Attenuates Pain Caused by Oxaliplatin in Mice.
      . Intraperitoneal (i.p.) or intrathecal (i.t.) injection with minocycline both alleviated cisplatin-induced mechanical allodynia and sensory deficits, and prevented IENFs loss
      • Hu LY
      • Zhou Y
      • Cui WQ
      • Hu XM
      • Du LX
      • Mi WL
      • Chu YX
      • Wu GC
      • Wang YQ
      • QL Mao-Ying
      Triggering receptor expressed on myeloid cells 2 (TREM2) dependent microglial activation promotes cisplatin-induced peripheral neuropathy in mice.
      . Also, a review of clinical studies on minocycline noted that it showed a positive pain-reducing effect to some of chemotherapy-induced neuropathic pain cases
      • Shin DA
      • Kim TU
      • Chang MC.
      Minocycline for Controlling Neuropathic Pain: A Systematic Narrative Review of Studies in Humans.
      . However, in a Phase II randomized trial, minocycline did not reduce patients’ CIPN symptoms such as numbness or fatigue
      • Wang XS
      • Shi Q
      • Bhadkamkar NA
      • Cleeland CS
      • Garcia-Gonzalez A
      • Aguilar JR
      • Heijnen C
      • Eng C.
      Minocycline for Symptom Reduction During Oxaliplatin-Based Chemotherapy for Colorectal Cancer: A Phase II Randomized Clinical Trial.
      . Therefore, further studies should be done to explain these contradictions. Whatever, with further research on the mechanism of CIPN, the role of minocycline, especially its regulatory mechanism of intracellular signaling pathways, will be gradually deepened, and it is expected to become a bridge for the study of neuron-glial crosstalk and will be widely used in clinical, which can be our future research directions.

      Conclusions

      The present study demonstrated a novel role of P2Y12 signaling in the regulation of cisplatin-induced pain hypersensitivity through IL-18 production, which is mediated by activation of the microglial SFK-p38 pathway in the spinal microglia. Blockade of P2Y12 signaling could be a plausible pharmaceutical therapy to prevent and relieve cisplatin-induced pain hypersensitivity.

      Disclosures

      This study was supported by the National Nature Science Foundation of China (81571066 to Wen Shen) and the Key Project of the Natural Science Foundation of Jiangsu Education Department (16KJA320002 to Wen Shen). The authors declare no conflict of interest regarding the publication of this paper.

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