IRF8 is crucial for the nicotine withdrawal-induced hyperalgesia in mice

2.1 Animals

Male C57BL/6 mice (6–8 weeks old) were purchased from Pengyue Animal Experimental Center (Jinan, China, certificate number SCXK, Shandong 20190003). The mice were housed in a specific pathogen-free facility of Liaocheng Medicine Inspecting Institute (Shandong, China) with a cycle of 12 h light/dark, 25 ± 1°C and free access to food and water ad libitum. The experimental protocols were approved by the Institutional Animal Care and Use Committee of the Liaocheng People’s Hospital (Shandong, China) and conducted according to the “Guidelines for the Care and Use of Laboratory Animals” by the National Institutes of Health. All efforts were made to minimize animal suffering and to reduce the number of mice used.

1. Ethical approval: The research related to animals use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. The research project basically conformed to international medical ethics documents and relevant Chinese laws, regulations and ethics requirements. The researchers had guaranteed that they abided by the principles stated by the World Medical Association and respected the ethical recommendations made by the ethics committee on this project. Ethical approval has been granted by Medical Ethics Committee of Liaocheng People’s Hospital affiliated to Shandong University, China (approval number: 2017053).

2.2 Lentivirus construction

Lentiviruses for the expression of control shRNA (NC-LV, 5′-TTCTCCGAACGTGTCACGT-3′) or IRF8-specific shRNA (IRF8-RNAi-LV, 5′-ATCCGAGAGCTGCAGCAATTC-3′, NM_008320) were generated by Shanghai Genechem (Shanghai, China). Both IRF8-RNAi-LV and NC-LV were pseudocoated with vesicular stomatitis virus G glycoprotein, which has been demonstrated to target and transduce glial cells effectively [19]. The generated IRF8-RNAi-LV and NC-LV had 2 × 10⁹ TU/mL and 8 × 10⁸TU/mL, respectively.

2.3 Model of NTW, treatment and collection of samples

We established a mouse model of NTW, as described previously [17] with minor modifications. Mice were injected subcutaneously with vehicle saline (Control) or 3 mg/kg nicotine (nicotine hydrogen tartrate salt, [−]-1-methyl-2[3-pyridyl]pyrrolidine [Sigma, St. Louis, USA]) in 0.9% normal saline] for three times per day (7 am, 3 pm, 11 pm) for 7 consecutive days. One hour after the last injection on the 7th day, the mice were injected subcutaneously with 1 mg/kg mecamylamine (Sigma) to trigger NTW. The Control and NTW groups (n = 20 per group) of mice were tested for their behaviors between in 8 am—and 10 am every day for 1, 4, 7, 10 and 14 days post NTW. After each behavioral assessment, three mice from each group were sacrificed, and their L_4–6 spinal cords were dissected for western blot. The remaining five mice in each group were sacrificed after day 7 behavioral assessment, and their L_4–6 spinal cord tissues were dissected for immunohistochemistry.

1 day after injection with mecamylamine, another 36 NTW mice were randomized and infused intrathecally with vehicle saline, NC-LV or IRF8-RNAi-LV at 8 am every day for three consecutive days [20]. Briefly, individual mice were anesthetized and inserted with a 30-gauge microinjection needle connected to a 10 µL Hamilton microsyringe into L4 or L5 spinous space at a 20-degree angle above the vertebrae column. When the need entered the subarachnoid space, the mice were infused with 5 µL saline (NS); the same volume saline containing 4 × 10⁶ TU NC-LV or IRF8-RNAi-LV, respectively. Their behavioral assessments were performed between 8 am and 10 am every day for 7 consecutive days post the last intrathecal infusion (n = 12 per group). Subsequently, the mice were sacrificed, and their L_4–6 spinal cord tissues were dissected for immunohistochemistry (n = 5 per group), western blot and quantitative RT-PCR (n = 7 per group).

2.4 Measurement of thermal hyperalgesia

Individual mice were tested for their thermal hyperalgesia using the BME-410C automatic thermal stimulus apparatus (Medical Science Biomedical Engineering Research, China), as a previous description [21]. Briefly, individual mice were acclimated on the hind paw plantar surface for 15 min and stimulated with heat using a radiant thermal stimulus (a 12 V/10 W halogen lamp with a stimulating light area of <20 mm² and timing accuracy of 10 ms) below the hind paw plantar surface. Each paw was tested for three times with an interval of 5 min, and individual paws were tested for a maximum of 20 s. The paw withdrawal latencies (PWLs) were recorded, and the mean values of PWLs were further analyzed.

2.5 Immunohistochemistry

After each behavioral assessment, the mice were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneal) and perfused transcardially with cold phosphate buffered saline (PBS, 0.1 M, pH 7.4), followed by ice-cold 4% paraformaldehyde in PBS. The lumbar spinal cords (L4–L6 segments) were immediately dissected and post-fixed in 4% paraformaldehyde at 4°C overnight, followed by paraffin-embedding. The tissue sections (−6 µm) were dewaxed, rehydrated and subjected to antigen retrieval. The sections were blocked with 5% bovine serum albumin and incubated with primary antibodies against IRF8 or Iba1 (rabbit anti-IRF8, orb86775, 1:50, Biorbyt, England; rabbit anti-Iba1, ab178847 1:800, Abcam, USA) at 4°C overnight. After being washed, the sections were incubated with biotinylated goat anti-rabbit IgG and SABC (Beyotime, Nanjing, Jiangsu, China), followed by reaction with DAB and counterstained with hematoxylin. The stained signals were captured under a light microscope (Olympus) and semiquantitatively analyzed. The number of IRF8+ cells and the solidity of Iba-1+ microglial cells in five fields of each sample were counted in a blinded manner. The stained intensity was analyzed by ImageJ (NIH, USA) and the solidity of microglia was analyzed, as previous description [22]. In general, the more ramified thinner microglia, the bigger convex area is, thus yielding a smaller solidity index as resting microglia.

2.6 Quantitative RT-PCR

The dissected fresh spinal cord tissues were frozen in liquid nitrogen and stored at −80°C until analysis. Total RNA was extracted from each spinal cord sample using the Trizol (Takara, Dalian, China) and reverse-transcribed into cDNA using a Reverse Transcription Kit (Takara). The relative levels of target gene to the control GAPDH mRNA transcripts were determined in triplicate by quantitative RT-PCR using a SYBR Premix Ex TaqTMII (Takara) and specific primers (Sangon Biotech) in an ABI 7500 (Applied Biosystems, USA). The data were analyzed by 2^−ΔΔCt. The sequences of primers were sense 5′-TGTTCGTGAAGCGGCTGTGC-3′ and antisense 5′-GCTCTCGGATGAACTGGTTGGTG-3′ for IRF8; sense 5′-CTCATCCTGGCTTACGTCATT-3′ and antisense 5′-GAATCCAAGCTGAGAAGTGTTG-3′ for P2RX4; sense: 5′-CCCATGAAAGAAGTAAACGTCC-3′ and antisense 5′-CCCATGAAAGAAGTAAACGTCC-3′ for BDNF; sense 5′-TGTTCGTGAAGCGGCTGTGC-3′ and antisense 5′-GCTCTCGGATGAACTGGTTGGTG-3′ for GAPDH.

2.7 Western blot

The frozen spinal cord tissue samples were homogenized in ice-cold lysis buffer containing a cocktail of protease inhibitors and phosphatase inhibitors (Sigma) and centrifuged. After measuring the protein concentrations in the lysate supernatants using BCA Protein Assay Kit (Beyotime Biotechnology), the lysates (15 µg/lane) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gels and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat dry milk in TBST and incubated overnight at 4°C with rabbit anti-IRF8 (LS-C335605, 1:600, Lifespan, USA), rabbit anti-P2RX4 (LS-C334869,1:800, Lifespan), rabbit anti-BDNF (28205-1-AP, 1:800, Proteintech, China) or mouse anti-β-actin (1:1,000, AA128, Beyotime). After being washed, the bound antibodies were detected with goat antirabbit or goat antimouse IgG/HRP secondary antibodies (1:1,000, Beyotime) and visualized with enhanced chemiluminescent reagents. The relative levels of target to β-actin protein expression were determined by densitometric analysis using ImageJ (NIH, USA) (Figure 1).

Figure 1

Experimental design and timelines. C57BL/6 mice were randomized and injected subcutaneously with saline or nicotine per day for 7 consecutive days, followed by injection with mecamylamine to induce NTW. Their thermal hyperalgesia were measured and the expression of IRF8 in the spinal cord tissues was determined. In addition, the NTW mice were randomized and infused intrathecally with vehicle saline (NS), control lentivirus (NC-LV), or lentivirus for the expression of IRF8-specific shRNA (IRF8-RNAi-LV) for three days. Their PWLs, microglia activation, IRF8, P2X4R and BDNF expression in the spinal cord tissues were determined.

2.8 Statistical analysis

Data are expressed as mean ± SD and graphed using Prism 6.0 (GraphPad, La Jolla, CA). The repeated measurements for PWL with multiple time points in each group were analyzed by two-way ANOVA and post-hoc Bonferroni’s test. Other quantitative data among the groups were analyzed by one-way ANOVA and post-hoc LSD test or Student’s t-test. All statistical analyses were performed using SPSS 16.0 statistical software. A p-value of <0.05 was considered statistically significant.

3.1 NTW decreases PWLs and increases IRF8 expression in the spinal cord tissues of mice

NTW has been associated with a decrease in thermal pain threshold [16,17,18]. To explore the molecular mechanisms underlying the effect of NTW, a mouse model of NTW was established. Examination of these mice indicated that there was no significant difference in the baseline of PWLs in the hind limb between the Control and NTW groups of mice (Figure 2a). In comparison with that in the Control, the PWLs were significantly decreased in the NTW group at 1 day post NTW (P < 0.05) and were the lowest in the NTW mice at 7 days post NTW (P < 0.01), followed by a gradual raise (Figure 2a). However, the PWLs in the NTW group at 14 days post NTW remained significantly lower than that in the Control (P < 0.05). These results clearly indicated that NTW significantly decreased thermal pain thresholds in mice, consistent with our previous studies [16,17,18].

Figure 2

NTW decreases PWLs and increases IRF8 expression in the spinal cord of mice. C57BL/6 mice were randomized and injected with saline as the Control or induced for NTW (n = 20 per group). Their thermal hyperalgesia was assessed longitudinally by PWLs after NTW. Some mice (n = 3 per group) were sacrificed, and their spinal cord tissues were dissected and tested for the expression of IRF8 by western blot. Some mice (n = 7 per group) from each group were sacrificed after behavioral test on day 7 post NTW, and the numbers of IRF8+ microglia in the spinal cord tissues were determined by immunohistochemistry. Data are representative images (magnification × 200) or expressed as the mean ± SD of each group from three separate experiments. (a) The PWLs. (b) Western blot analysis of IRF8 expression. (c) Immunohistochemistry analysis of IRF8+ cells in the spinal cord tissues. *P < 0.05, **P < 0.01 versus the control group. ^##P < 0.01 versus the day 4 time point.

Because IRF8 is related to microglia activation and pain sensitivity [13,14], we began to explore the potential mechanisms underlying the pathogenesis of NTW-related hyperalgesia by testing the relative levels of IRF8 expression in the spinal cord tissues of mice. Western blot analysis revealed that the relative levels of IRF8 expression in the spinal cord tissues of the NTW group were significantly upregulated on day 1, 4, 7, 10, or 14 post NTW, compared with that in the Control mice (P < 0.05 or P < 0.01, Figure 2b). Interestingly, the highest levels of IRF8 expression appeared on days 4 and 7 post NTW in mice. Consistently, immunohistochemistry indicated that the numbers of IRF8+ cells in the spinal cord tissues on day 7 post NTW were significantly greater than that in the Control mice (P < 0.01, Figure 2c). The levels of IRF8 expression in the spinal cord tissues were negatively associated with the PWLs in mice, suggesting that increased IRF8 expression may contribute to decreased thermal PWLs in the NTW group of mice.

3.2 IRF8-silencing attenuates thermal hyperalgesia in the NTW mice

To further confirm the role of IRF8 in NTW-induced hyperalgesia, we tested whether knockdown of IRF8 expression in the spinal cord tissues could mitigate the NTW-induced hyperalgesia in mice. First, we generated lentiviruses for the expression of IRF8-specific shRNA (IRF8-RNAi-LV) or control RNA (NC-LV). The NTW mice were randomized and infused intrathecally with vehicle saline (NS), control NC-LV or IRF8-RNAi-LV every day for three consecutive days, and their PWLs were tested daily up to 7 days post the last virus infusion. There were similar levels of PWLs between the NS and NC-LV groups of mice, indicating that infusion with NC-LV did not affect the NTW-induced hyperalgesia in mice (Figure 3a). Compared with the NC-LV or NS groups, intrathecal infusion with IRF8-RNAi-LV significantly increased PWLs in the NTW mice on day 4 post the last virus infusion (P < 0.01, Figure 3a) and continually prolonged up to 7 days post virus infusion in the NTW mice. In addition, we found that the relative levels of IRF8 expression in the spinal cord tissues of the IRF8-RNAi-LV group of mice were significantly reduced by about 27% compared with other two groups of mice (P < 0.01, Figure 3b). Similarly, IRF8-silencing also significantly decreased the relative levels of IRF8 mRNA transcripts in the spinal cord tissues of the NTW mice (P < 0.01, Figure 3c). Thus, IRF8-silencing mitigated the NTW-induced thermal hyperalgesia in mice.

Figure 3

Intrathecal administration with lentivirus (IRF8-RNAi-LV) for IRF8-specific shRNA expression, but not the control NC-LV, attenuates the NTW-induced hyperalgesia in mice. One day after NTW, the mice were randomized and infused intrathecally with vehicle saline, NC-LV or IRF8-RNAi-LV every day for three consecutive days. Their behavioral assessments were performed daily for 7 consecutive days post the last intrathecal infusion (n = 12 per group). Subsequently, the mice were sacrificed and their L_4–6 spinal cord tissues were dissected, and the relative levels of IRF8 expression were determined by western blot and quantitative RT-PCR (n = 7 per group). Data are representative images (magnification × 200) or expressed as mean ± SD of each group from three separate experiments. (a) The PWLs. (b) Western blot analysis of IRF8 expression in the spinal cord tissues. (c) Quantitative RT-PCR analysis of IRF8 mRNA transcripts. **P < 0.01 versus the NS group.

3.3 IRF8-silencing mitigates the NTW-stimulated microglia activation and P2X4R and BDNF expression in the spinal cord tissues of mice

Our previous study has shown that NTW increases microglial cell activation and upregulates P2X4R and BDNF expression in the lumbar spinal cord of rats [18]. To understand the mechanisms underlying the action of IRF8-silencing in attenuating thermal hyperalgesia in mice, we tested whether IRF8-silencing could modulate the NTW-stimulated microglial cell activation, P2X4R and BDNF expression in mice. Immunohistochemistry analysis revealed that there were many microglial cells with a bigger body and shorter and thicker processes, the hallmarks of activated microglia in the spinal cord tissues of the NS and NC-LV groups of mice while many microglial cells displayed smaller body with longer and thinner processes, and the characteristics of resting microglia in the spinal cord tissues of the IRF8-RNAi-LV group of mice (Figure 4a). Quantitative analysis indicated that the solidity index of microglia in the spinal cord tissues of the IRF8-RNAi-LV group of mice was significantly less than that of the NS and NC-LV groups of mice (P < 0.01, Figure 4a).

Figure 4

IRF8-silencing mitigates the NTW-enhanced microglia activation, P2X4R and BDNF expression in the spinal cord tissues of mice. On day 7 post the last virus infusion, the different groups of mice were sacrificed and their spinal cord tissues were dissected. (a) Immunohistochemistry analysis of microglia using anti-Iba1 and quantification of the solidity of microglia in the spinal cord tissues. (b) Western blot analysis of P2X4R and BDNF expression. (c) Quantitative RT-PCR analysis of P2X4R and BDNF mRNA transcripts in the spinal cord tissues of mice. Data are representative images (magnification ×200) or expressed as mean ± SD of each group (n = 5 per group) from three separate experiments. **P < 0.01 versus the NC-LV group.

Western blot analysis revealed that the relative level of P2X4R and BDNF protein expression in the spinal cord tissues of the IRF8-RNAi-LV group was significantly reduced by 50–62%, compared with that in the NS and NC-LV groups of mice (P < 0.01, Figure 4b). A similar pattern was detected for P2X4R and BDNF mRNA transcripts among groups of mice (Figure 4c). Collectively, such data demonstrated that decreased IRF8 expression significantly mitigated the NTW-stimulated microglial cell activation and P2X4R and BDNF expression in the spinal cord tissues of mice.