Male Sprague-Dawley rats (280–320 g) were provided by the Laboratory Animal Center of the Fourth Military Medical University (FMMU). This study was carried out in accordance with the recommendations of the Animal Care and Use Committee of FMMU and performed in accordance with the updated Guide for the Care and Use of Laboratory Animals (8th edition, the National Academies Press, 2011). The number of rats used and their suffering were minimized. During the whole experiment, the rats were randomized.

Surgery was performed according to the methods described previously (Yang et al., 2014; Yang Y. et al., 2016). Rats were anesthetized with chloral hydrate (0.3 g/kg, i.p.) and placed in a stereotaxic apparatus (Narishige Scientific Instrument Lab, Japan). Collagenase type IV (0.025 U/0.25 μl, Sigma-Aldrich China, Shanghai) or saline (0.25 μl) was microinjected into the ventral posterior lateral nucleus (VPL) of the right thalamus (bregma −3.48 mm antero-posterior; 3.6 mm lateral to the midline, and 6.2 mm ventral to the brain surface) according to the stereotaxic coordinates (Paxinos and Watson, 2005). After each injection, the syringe remained for 5 min to prevent spread of the agent to the brain surface. Then the needle was slowly withdrawn, the skin closed using 4.0 sutures, and all rats were allowed to recover in individual cages for at least 3 days. Naïve rats were fed under the same conditions in a parallel manner.

The mechanical pain sensitivity testing was performed by experimenters blinded to the experimental treatments (Yang et al., 2014; Yang Y. et al., 2016). The rats were placed on a metal mesh floor and habituated for 1 h before testing. Ascending graded von Frey filaments were applied from underneath the metal mesh floor to the plantar area of the appropriate hindpaw. Each von Frey filament was applied 10 times (once every several seconds). The bending force value of the von Frey filament that caused an appropriate 50% occurrence of paw withdrawal was expressed as the paw withdrawal mechanical threshold (PWMT, g). Baseline PWMT of all experimental rats were measured prior to the surgery and the effects of different drugs on PWMT were evaluated at the time point indicated in corresponding figures.

Intra-thalamic drug injections were performed at 10 days after collagenase injection when CPSP was stably established or at 30 min before collagenase injection prior to CPSP induction. The rats were injected with minocycline hydrochloride (Sigma, 10 μg), fluorocitrate (Sigma, 1 nM), AMD3100 (Abcam, 7.5 or 15 μg) or YC-1 (Abcam, 0.2 mM) in a total volume of 1 μl over a period of 10 min. The doses used in the present study were based on or slightly modified from previous studies (Zhao et al., 2007; Trecki and Unterwald, 2009; Huang et al., 2014; Luo et al., 2014; Choi et al., 2015). Minocycline hydrochloride selectively inhibits microglial activation and proliferation, with no direct effect on neurons or astrocytes. Fluorocitrate, an inhibitor of astrocyte metabolism, inhibits the tricarboxylic acid cycle enzyme aconitase in astrocytes. AMD3100, which is a highly selective CXCR4 antagonist, was used to evaluate the roles of CXCR4 in the development of CPSP. YC-1, an established HIF-1α inhibitor, was applied to verify the regulatory effects of HIF-1α in the development of CPSP. A separate group of naïve rats underwent intra-thalamic injection of recombinant active SDF1 protein (Abcam, 50 ng) or CoCl₂ (Sinopharm Chemical Reagent Co. Ltd, 100 mM), a well-known inducer of HIF-1α, following minocycline, fluorocitrate or AMD3100 injections to explore the cellular mechanisms underlying the pro-algesic effect of SDF1-CXCR4 signaling. The intra-thalamic injection methods were the same as above-mentioned and the injection locations were assessed histologically. Fluorocitrate was dissolved in 0.3% 2 M HCl in phosphate-buffered saline (PBS) and YC-1 was dissolved in 1% dimethyl sulfoxide (DMSO), whereas all other drugs were dissolved in sterile saline.

As reported previously (Yang et al., 2015), the rats were anesthetized with chloral hydrate, then perfused with physiological saline, followed by 4% paraformaldehyde in 0.1 M PB solution. The brain was removed and postfixed overnight at 4°C and then immersed in 30% sucrose in 0.1 M PB. Brain tissue were cut into transverse sections (30 μm thick) on CM1900 freezing microtome (Leica, Germany). After blocking with 10% goat serum in PBS, the sections were incubated overnight at 4°C with the following primary antibodies: mouse anti-GFAP (1:500 Millipore), rabbit anti-Iba-1 (1:500, WAKO), mouse anti-NeuN (1:200, Millipore), Goat anti-CXCR4 (1:200, Abcam) and rabbit anti-c-Fos (1:500, Abcam). On the following day, Cy3- or FITC-conjugated secondary antibodies were incubated for 2–3 h at room temperature. For double immunostaining, sections were incubated with a mixture of primary antibodies overnight at 4°C, followed by a mixture of secondary antibodies. The images were examined under a laser scan confocal fluorescent microscope (Olympus FV1000, Japan).

As reported previously, the thalamus tissues around the hemorrhagic lesion sites were collected and homogenized in RIPA lysis buffer containing protease inhibitors (Applygen Technologies Inc., China; Yang et al., 2015, 2017; Yang Y. et al., 2016). Protein concentrations of the lysate were determined using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein were separated by 10% separation gels and then transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% nonfat milk (Bio-Rad, Hercules, CA, USA) for 1 h at room temperature, the membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies included mouse anti-HIF-1α (1:500, Abcam), rabbit anti-SDF1 (1:200, Abcam) and the antibodies mentioned in immunohistochemistry section. On the following day, the membranes were washed three times in PBST and then incubated with an HRP-conjugated secondary antibody (Bio-Rad) for 2 h at room temperature. The membranes were visualized with enhanced chemiluminescence solution (Alpha Innotech Corp) and the signals were captured with FluorChem FC2 (Alpha Innotech Corp). The density of specific bands was measured with a computer-assisted imaging analysis system and normalized to β-tubulin or β-actin intensity.

The thalamus tissues around the hemorrhagic lesion sites were collected and homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors (Applygen Technologies Inc.). After quantitative measurement of the total protein concentrations with a BCA protein assay kit (Thermo Scientific), the homogenized thalamus tissue were assayed for TNF-α, IL-1β and IL-6 (Westang, Shanghai, China) by ELISA. Each protein of interest was expressed as picograms per milligram of total proteins.

Data were analyzed using GraphPad Prism version 5 (GraphPad, San Diego, CA, USA) and all data were expressed as means ± SEM. Differences in changes of values of each group were tested using t-tests and one-way or two-way repeated ANOVA, followed by individual post hoc comparisons (Bonferroni or Tukey test). Linear relationships were assessed using Pearson’s correlation test. A level of P < 0.05 was accepted as significant.

Similar to our previous reports (Yang et al., 2014), unilateral ITC injection confined to the VPL thalamic nucleus (Figure 1A) resulted in bilateral reductions in PWMT, which were identified on day 7 post-injection and remained unchanged until day 28 post-injection, suggesting a chronic, persistent bilateral mechanical allodynia in this model (Figure 1B). To examine the involvement of microglia and astrocytes, the expression of Iba-1 and GFAP in the peri-thalamic lesion (hematoma) sites were quantified by immunohistochemistry and Western blot on 3, 7, 14 and 28 days after ITC. Compared with intra-thalamic saline (ITS) injection group, the ITC group showed marked increases in Iba-1 and GFAP expression at each time point examined. The expression level of Iba-1 and GFAP was substantially increased in peri-thalamic lesion sites on day 3 after ITC, reached peak on day 7 and remained unchanged until day 28 (Figures 1C,D). However, the expression level of Iba-1 and GFAP in the contralateral thalamus remained at basal levels (Figure 1).

After CPSP was well established by 10 days after ITC, intra-thalamic injection of minocycline selectively blocked the upregulation of Iba-1 but without any effect on GFAP expression in the peri-thalamic lesion sites, while intra-thalamic injection of fluorocitrate significantly diminished ITC-induced activation of astrocytes labeled by GFAP but without any effect on Iba-1 expression (Figures 2A,B). To investigate the roles of microglial and astrocytic activation in the CPSP, we explored the time-related effects of minocycline and fluorocitrate on ITC-induced bilateral mechanical pain hypersensitivity. Our results showed that single intra-thalamic injection with minocycline alleviated the established bilateral mechanical pain hypersensitivity. The anti-allodynic effect of minocycline reached peak at 6 h after injection and maintained at a significant level for at least 7 days. The established bilateral mechanical pain hypersensitivity was also remarkably reduced by fluorocitrate, which lasted from 4 h to 3 days after injection (Figure 2C). All these results suggest that both microglia and astrocytes play important roles in maintaining the thalamic hemorrhage-induced CPSP.

As SDF1-CXCR4 signaling was closely related to microglia and astrocyte activation, we next tried to determine whether SDF1-CXCR4 signaling was involved in the induction of pain in naïve rats through regulating glial cell activation. As shown in Figure 3A, intra-thalamic SDF1 injection elicited bilateral mechanical pain hypersentivity from day 3 to day 7 which could be completely blocked by pre-administration of AMD3100 into the SDF1 injection sites. Moreover, pre-administration of minocycline or fluorocitrate in the SDF1 injection sites also significantly suppressed SDF1-induced bilateral mechanical pain hypersentivity from day 5 to day 7 (Figure 3B).

Under the CPSP condition induced by ITC, SDF1 expression was significantly increased in the peri-thalamic lesion sites at 3, 7, 14 and 28 days post-ITC relative to the saline control (Figure 4A). The level of CXCR4 expression in peri-thalamic lesion sites under CPSP condition was also examined by Western blot analysis. As shown in Figure 4B, increased CXCR4 protein expression was also seen at 3, 7, 14 and 28 days post-ITC relative to the saline control.

To define the cellular distribution of CXCR4, double staining of CXCR4 with different cell markers was performed. CXCR4 was not only co-localized with neuronal marker NeuN but also with astrocytic marker GFAP and microglial marker Iba-1 (Figure 4C).

To determine whether CXCR4 contributes to microglial and astrocytic activations under CPSP condition, intra-thalamic VPL injection of AMD3100 (15 μg) was administered 30 min prior to ITC. As shown in Figure 5A, CXCR4 antagonism prevented increases in both Iba-1 and GFAP expression in the peri-thalamic lesion sites on day 3 and 7 post-ITC. Moreover, the increased release of pro-inflammatory mediators including TNF-α, IL-6 and IL-1β in the peri-thalamic lesion sites was also prevented by intra-VPL AMD3100 administration on day 7 and 14 post-ITC (Figure 5B). Furthermore, antagonizing CXCR4 by intra-thalamic AMD3100 on day 10 post-ITC also suppressed the increase in Iba-1 and GFAP expressions, implicating that SDF1-CXCR4 signaling is directly involved in the ITC-induced activations of both microglia and astrocytes (Figures 5C–E).

The role of CXCR4 in the neuronal activity was also examined by measuring c-Fos expression surrounding the glial barrier. As shown in Figure 5F, the c-Fos immunoreactivity was barely observed in rats receiving ITS, while significant increase in c-Fos-labeled neurons was observed in the peri-thalamic lesion sites following ITC. Intra-thalamic microinjection of AMD3100 reduced the number of c-Fos-labeled neurons (Figure 5F) and decreased the ITC-induced c-Fos expression as measured by Western blot (Figure 5G).

To determine whether CXCR4 contributes to the induction of thalamic hemorrhage-induced CPSP, AMD3100 (15 μg) was microinjected into the VPL 30 min prior to ITC. Our behavioral assay verified that pre-injection of AMD3100 partially inhibited the ITC-induced bilateral mechanical pain hypersensitivity for 21 days relative to ITS control (Figure 6A), implying that CXCR4 plays a partial role in the initiation of CPSP.

However, the maintaining role of CXCR4 in the thalamic hemorrhage-induced CPSP was more significant. As shown in Figure 6B, intra-thalamic injection of AMD3100 with two doses (7.5 μg and 15 μg) on day 10 after ITC almost completely reversed the well-established CPSP. The analgesic effect of AMD3100 was dose-related and lasted for 48 h to 72 h, implicating the therapeutic value of AMD3100 in treatment of CPSP (Figures 6B,C).

In the present study, using Western blot analysis (Figure 7A), we examined HIF-1α expression in the peri-thalamic lesion sites at 3, 7, 14 and 28 days after ITC. It was revealed that ITC resulted in a short-term increase in HIF-1α expression, reaching peak during day 3 and 7, returning to the control level since day 14 post-ITC (Figure 7A).

We next explored whether HIF-1α is involved in the development of ITC-induced CPSP. It was interesting to find that intra-thalamic microinjection of YC-1, a potent inhibitor of HIF-1α production, blocked the occurrence of ITC-induced CPSP when being administered 30 min prior to ITC (Figure 7B), however it had no reversal effect on the well-established CPSP when being administered 10 days after ITC (Figure 7C).

To verify whether HIF-1α is directly involved in regulating the expression of SDF1-CXCR4 signaling under CPSP condition, intra-thalamic injection of YC-1 was administered 30 min prior to ITC. Western blot results showed that, similar to CXCR4 antagonism by AMD3100, local inhibition of HIF-1α expression by YC-1 resulted in significant reduction in the expression of both SDF1/CXCR4 (Figures 8A,B) and Iba-1/GFAP (Figures 8A,C) on day 3 and day 7 post-ITC.

Intra-thalamic injection of CoCl₂, a well-known inducer of HIF-1α, was shown to result in elevation of HIF-1α, SDF1 and CXCR4 expressions examined on day 3 and 7 post-injection (Figures 9A,B). Pre-blockade of HIF-1α production by YC-1 through intra-thalamic injection prevented the enhancement of HIF-1α, SDF1 and CXCR4 expression, whereas, pre-blockade of CXCR4 with AMD3100 failed to change the enhanced expression of HIF-1α although SDF1 and CXCR4 up-regulation was prevented (Figures 9A,B). Similar to the results of ITC, intra-thalamic injection of CoCl₂ induced overexpression of Iba-1 and GFAP (Figures 9A,C), suggesting activation of both microglia cells and astrocytes by CoCl₂. The CoCl₂-induced overexpression of SDF1 and CXCR4 and activations of microglia and astrocytes were also blocked by pre-injection of AMD3100 or YC-1, respectively (Figures 9A–C).

Similar to the results of ITC, intra-thalamic injection of CoCl₂ induced bilateral mechanical pain hypersensitivity that could be blocked by pre-treatment of AMD3100 through intra-thalamic microinjection (Figure 9D).

HIF-1α is a key transcription factor for the adaptive responses to hypoxia (Singh et al., 2012). In the present study, we found that the protein level of HIF-1α was significantly increased at the early phase, but not in the late phase, after hemorrhagic stroke induced by ITC. As supporting evidence, Jiang et al. (2002) reported that HIF-1α was significantly up-regulated in the basal ganglia following experimental intracerebral hemorrhage in rats receiving injections of autologous blood, thrombin, or lysed red blood cells. Here, we further found that inhibition of HIF-1α could prevent the development of CPSP but without analgesic effect during the maintenance of CPSP, implicating that up-regulation of the downstream gene expression of HIF-1α is initiated by HIF-1α but maintained by other mechanisms. By utilizing YC-1, the most specific inhibitor available for HIF-1α (Zhang et al., 2014), it was demonstrated that the increased expression of SDF1 and CXCR4 in the peri-thalamic lesion sites was almost completely suppressed. In fact, it has been demonstrated that both SDF1 and CXCR4 promoters contain hypoxia response elements (HREs) which have a HIF-1 DNA binding site (Hitchon et al., 2002; Schioppa et al., 2003; Speth et al., 2014) and blockade of HIF-1α activation can abrogate both hypoxia-induced CXCR4 up-regulation and SDF1-dependent cell migration in microglia in vitro (Wang et al., 2008). In the current study, the thalamic hemorrhage-induced microglial and astrocytic activations was prevented by blockade of both HIF-1α and SDF1-CXCR4 signaling, suggesting that HIF-1α plays a critical role in the up-regulation of SDF1-CXCR4 signaling that subsequently mediates the glial cell activations at the late stage of thalamic hemorrhage condition. To further verify the role of HIF-1α, we injected CoCl₂, a well-known inducer of HIF-1α, into the VPL of normal rats. As expected, the expression of SDF1 and CXCR4 protein was up-regulated by intra-VPL CoCl₂ microinjection and which was prevented by pre-inhibition of HIF-1α. Likewise, microglia and astrocytes was also activated by intra-VPL CoCl₂ microinjection and the activation could be blocked by pre-treatment with CXCR4 inhibitor, indicating that HIF-1α is essential for the induction of SDF1-CXCR4 signaling-mediated glial cell activations. This result can be supported by a previous report showing that both astrocytes and microglia could be activated by microinjection of CoCl₂ into the frontoparietal cortex of rats (Caltana et al., 2009). Most interestingly, our present study provided a new line of experimental evidence demonstrating that intra-thalamic CoCl₂ microinjection in normal rats is sufficient to induce transient bilateral mechanical pain hypersensitivity through HIF-1α activation. HIF-1α has also been shown to be involved in the development of peripheral neuropathic pain, demonstrated by HIF-1α gene knockout in primary nociceptive neurons (Kanngiesser et al., 2014). Moreover, inhibiting accumulation of HIF-1α in the sciatic nerve also relieved both spared nerve injury-evoked and chronic constriction injury-induced neuropathic pain (Hsieh et al., 2012). However, none of these two previous studies clearly identified how HIF-1α worked to produce pain. Thus the link between up-regulation of SDF1-CXCR4 signaling by HIF-1α activation and the development of CPSP in the current study would shed new light on the treatment of CPSP in clinic.

To determine the roles of microglia and astrocytes in the maintenance of CPSP, we evaluated the analgesic effect of minocycline, a microglial inhibitor, and fluorocitrate, an astrocytic inhibitor, by intra-thalamic microinjection of these agents, respectively. As expected, both minocycline and fluorocitrate reversed the well-established mechanical pain hypersensitivity induced by ITC-induced hemorrhage, indicating involvement of activated microglial and astrocytic cells in the maintenance of CPSP. This result can be supported by a previous study showing attenuation of thalamic hemorrhage-induced mechanical allodynia following post-treatment with systemic minocycline (Hanada et al., 2014). Also, another line of evidence demonstrated that intra-thalamic microinjection of minocycline was sufficient to reverse sciatic nerve injury-induced thermal hyperalgesia for 30 min (LeBlanc et al., 2011). However, in an animal model of the spinal cord injury-induced mechanical pain hypersensitivity, it was shown that intra-thalamic minocycline microinjection produced anti-hyperalgesic effect for 8 h (Zhao et al., 2007). Herein, our results showed the duration of the analgesic effect of minocycline on CPSP was much longer and lasted at least for 7 days. The discrepancy in duration of analgesia for minocycline might be duo to various reasons such as differences in the time course of microglial cell activation in different pain models, drug delivery route and other unknown mechanisms involved. Unlike the sciatic nerve and spinal cord injury, thalamic hemorrhage was the primary injury in CPSP model and glial activation in the thalamus would be the critical underlying mechanism for CPSP which was the reason for us to adopt intra-thalamic injection in the present study. It has been already shown that the activated glial cells of both microglia and astrocytes can produce and release various types of chemokines and cytokines that not only trigger neuronal hyperexcitability but also facilitate inter-glial cell activations through positive feedback regulation (Guo et al., 2007; Sheridan and Murphy, 2013; Macht, 2016). Here we further demonstrated that chemokine SDF1 and its cognate receptor CXCR4 were both up-regulated in the peri-thalamic lesion sites after ITC and CXCR4 was localized not only in neurons but also in astrocytes and microglia. In line with previous studies, the temporal pattern of SDF1 up-regulation was similar to the up-regulation of GFAP and Iba1, suggesting that activated microglia and astrocytes are the main SDF1-producing cells in the peri-thalamic lesion sites (Shen et al., 2014; Bai et al., 2016; Yang L. et al., 2016). Based on the cellular localization of CXCR4 in both neurons and glial cells and the abundant production of SDF1 from activated astrocytes and microglia, we propose that through positive feedback regulation, SDF1-CXCR4 signaling would play critical roles in mediating microglial-astrocytic-neuronal interactions, because selectively blocking CXCR4 with intra-thalamic AMD3100 microinjection could: (1) remarkably suppress thalamic hemorrhage-induced microglia and astrocyte activation; (2) significantly inhibit the release of pro-inflammatory mediators such as TNF-α, IL-1β and IL-6 from activated glial cells; and (3) reverse thalamic hemorrhage-induced increase in neuronal activities labeled with c-Fos in the peri-thalamic lesion sites. The persistent release of pro-inflammatory mediators could cause neuronal hyperexcitability and sensitization through binding to their respective receptors (Zhang et al., 2011; Clark et al., 2013; Chirila et al., 2014). As a new line of evidence, we have previously demonstrated that blocking CXCR4 with AMD3100 directly inhibited the hyperexcitability of primary nociceptor neurons under peripheral inflammatory pain state (Yang et al., 2015). Thus, it is conceivable that SDF1-CXCR4 signaling could mediate microglial-astrocytic-neuronal interactions (crosstalk) through two possible mechanisms: (1) glial-neuronal interactions mediated by direct binding of astrocyte/microglia-derived SDF1 with neuronal CXCR4; and (2) positive feedback glial-glial interactions through direct binding of astrocyte- or microglia-derived SDF1 with glial CXCR4 (Figure 10). The glial-glial interactions may serve as the source of pro-inflammatory mediators that in turn cause neuronal hyperexcitability and sensitization.

Given the potential role of SDF1-CXCR4 signaling in the glial-glial interactions and glial-neuronal crosstalk, we here demonstrated that pre-treatment with CXCR4 inhibitor AMD3100 could prevent the development of CPSP accompanying with the reduction of neuroinflammation which was essential for CPSP. To further confirm whether SDF1-CXCR4 signaling participates in the CPSP process, we injected exogenous SDF1 into VPL of naïve rats. As expected, single intra-thalamic SDF1 injection was sufficient to induce bilateral mechanical pain hypersensitivity through CXCR4 agonism. In addition, the SDF1-induced pain hypersensitivity was prevented by inhibiting microglial and astrocytic activation. It was interesting to note that both the analgesic effect of intra-thalamic AMD3100 and hyperalgesic effect of intra-thalamic SDF1 remained for a relative longer duration after administration although AMD3100 and SDF1 themselves have a short-term half-life (Hendrix et al., 2004; Kirkpatrick et al., 2010). Why? One possibility is that SDF1-CXCR4 signaling serves as a priming signal to mediate a positive feedback between glial-glial interactions under CPSP state. Once being activated, the downstream inflammatory cascades (TNF-α, IL-1β and IL-6) would be recruited to promote the local persistent neuroinflammation in the lesioned thalamus (Figure 10). This was supported by a previous study showing that intra-thalamic injection of CCL21 induced significant microglia activation and pain hypersensitivity which lasted longer than its own half-life and intra-thalamic injection of minocycline could also suppress CCL21-induced changes (Zhao et al., 2007). Moreover, here we also showed that post-treatment with AMD3100 could reverse the established CPSP in a dose-related manner. Taken all the existing data into account, we conclude that SDF1-CXCR4 signaling-mediated glial-glial/glial-neuronal communications are required for the development and maintenance of CPSP. Since it has been demonstrated that treatment with P2X7 receptor antagonist and IL-1β antibody could also reduce thalamic hemorrhage-induced microglia aggregation, neuronal excitability and allodynia phenomena in CPSP rats (Kuan et al., 2015), a common upstream regulation by HIF-1α-SDF1-CXCR4 signaling might be shared.

Because the analgesic effect of intra-thalamic injection of lidocaine on the thalamic hemorrhage-induced tactile hypersensitivity is short-lasting (Gritsch et al., 2016), the use of AMD3100 (also named as plerixafor) to treat CPSP would be more promising. Its safety and efficacy was approved by the U.S. Food and Drug Administration in 2008 for the mobilization of hematopoietic stem cells (Hummel et al., 2014). Moreover, several other CXCR4 antagonists are currently in clinical trials for cancer, HIV and WHIM syndrome treatment, highly supporting its potential use for the treatment of CPSP in clinic (Debnath et al., 2013). Since intraperitoneal delivery of AMD3100 has been demonstrated to be effective in suppression of pain (Dubovy et al., 2010; Xie et al., 2016), SDF1-CXCR4 signaling pathway as a new therapeutic target for the treatment of CPSP might be promising.