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. Author manuscript; available in PMC: 2018 Mar 1.
Published in final edited form as: J Neurochem. 2017 Feb 1;140(6):963–976. doi: 10.1111/jnc.13952

Chronic pain and impaired glial glutamate transporter function in lupus-prone mice are ameliorated by blocking CSF-1 receptors

Xisheng Yan 1,2, Dylan W Maixner 1, Fen Li 3, Han-Rong Weng 1,*
PMCID: PMC5339048  NIHMSID: NIHMS842688  PMID: 28072466

Abstract

Systemic lupus erythematosus (SLE) is a multi-organ disease of unknown etiology in which the normal immune responses are directed against the body’s own healthy tissues. Patients with SLE often suffer from chronic pain. Currently, no animal studies have been reported about the mechanisms underlying pain in SLE. In this study, the development of chronic pain in MRL lupus-prone (MRL/lpr) mice, a well-established lupus mouse model, was characterized for the first time. We found that female MRL/lpr mice developed thermal hyperalgesia at the age of 13 weeks, and mechanical allodynia at the age of 16 weeks. MRL/lpr mice with chronic pain had activation of microglia and astrocytes, over-expression of CSF-1 and IL-1β, as well as suppression of glial glutamate transport function in the spinal cord. Intrathecal injection of either the CSF-1 blocker or IL-1 inhibitor attenuated thermal hyperalgesia in MRL/lpr mice. We provide evidence that the suppressed activity of glial glutamate transporters in the spinal dorsal horn in MRL/lpr mice is caused by activation of the CSF-1 and IL-1β signaling pathways. Our findings suggest that targeting the CSF-1 and IL-1β signaling pathways or the glial glutamate transporter in the spinal cord is an effective approach for the management of chronic pain caused by SLE.

Keywords: MRL/lpr, glutamate uptake, neuroinflammation, nociceptive, nociception

Graphical abstract

Patients with Systemic lupus erythematosus (SLE) often suffer from chronic pain. In this study we for the first time characterized the development of chronic pain in the lupus prone animal model. Our findings suggest that targeting the CSF-1 signaling pathway or the glial glutamate transporter in the spinal cord is an effective approach for the management of chronic pain caused by SLE.

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Introduction

Systemic lupus erythematosus (SLE) is a multi-organ disease of unknown etiology in which the normal immune responses are directed against the body’s own healthy tissues. Although advances in medical management have greatly reduced mortality associated with SLE, pain still occurs in up to 90% of SLE patients, and significantly impairs their quality of life and productivity (Grigor et al., 1978, Hochberg and Sutton, 1988, Waldheim et al., 2013). While pain is a significant symptom during the active stage of SLE, studies also show that severe pain can occur in patients with mild and moderate SLE disease activities (Greco et al., 2003, 2004). Currently, no animal studies have been conducted to investigate the mechanisms underlying pain in SLE in animal models. Identifying signaling molecules regulating the generation of chronic pain in SLE will provide strategies for the treatment and development of analgesics for pain in SLE patients.

The MRL lupus-prone (MRL/lpr) mouse is a well-established mouse model of human SLE (Theofilopoulos and Dixon, 1985, Perez de Lema et al., 2001, Reilly and Gilkeson, 2002, Larson et al., 2012, Kattah et al., 2015). MRL/lpr mice, which carry a mutation in the apoptosis-related Fas gene, spontaneously develop a severe autoimmune disease with many features similar to those in SLE patients, including immune abnormalities affecting T and B cells, autoantibody production, immune complex formation, and systemic inflammation (Theofilopoulos and Dixon, 1985, Reilly and Gilkeson, 2002). As manifested in SLE patients, MRL/lpr mice exhibit inflammation in multiple tissues (organs) such as joints (Greco et al., 2003, Yoneda et al., 2004, Grossman, 2009, Cox et al., 2010), muscles (Edwards et al., 1986), vasculature (Yamada et al., 2003), and kidneys (Andrews et al., 1978, Yen et al., 2013). Chronic pain in MRL/lpr mice remains to be elucidated.

It is generally believed that pathological pain is caused by aberrant neuronal activation along the pain signaling pathway including peripheral sensory neurons, neurons in the spinal dorsal horn and supra-spinal areas. Increased neuronal activity at the spinal dorsal horn (spinal central sensitization) contributes importantly to the generation of pathological pain induced by nerve injury (Yan and Weng, 2013) or inflammation in peripheral tissue (Liu et al., 2008, Ren and Dubner, 2010, Ji et al., 2013). We and others have demonstrated that enhancement of excitatory glutamatergic synaptic activities is an important synaptic mechanism underlying the increased neuronal activities at the spinal dorsal horn in animals with neuropathic pain induced by nerve injury(Yan and Weng, 2013), or inflammatory pain induced by complete Freund’s adjuvant (CFA) (Meng et al., 2013). Glutamatergic synaptic activities are governed by three major factors: the amount of glutamate released from presynaptic neurons, the function of glutamate receptors at postsynaptic neurons, and the clearance of glutamate (Danbolt, 2001). As extrasynaptic glutamate cannot be metabolized extracellularly, clearance and homeostasis of extrasynaptic glutamate depend on glutamate transporters located in astrocytes and neurons (Danbolt, 2001). It is known that majority of glutamate uptake are carried by glial glutamate transporters (Haugeto et al., 1996). Impairment of glial glutamate transporters in the spinal dorsal horn enhances activation of AMPA and NMDA receptors in the spinal dorsal horn and contributes importantly to the excessive activation of spinal dorsal horn neurons in rats with neuropathic pain (Weng et al., 2007, Nie and Weng, 2009, 2010). Currently, it is unknown whether and how glial cells are implicated in the generation of chronic pain in SLE.

Numerous studies have demonstrated that activation of microglia and astrocytes and their subsequent release of inflammatory mediators play a critical role in the genesis of chronic pain induced by nerve injury (Ji et al., 2014), arthritis(Nieto et al., 2016), or inflammation induced by CFA (Ren and Dubner, 2010). Colony-stimulating factor-1 (CSF-1) is a cytokine that exerts its action by binding to the CSF- 1 receptor (CSF-1R), a type III receptor tyrosine kinase (Pixley and Stanley, 2004). CSF-1 is a major regulator for the survival, proliferation, and differentiation of mononuclear cells, macrophage, and microglia (Chitu et al., 2016). Recent studies show that upregulation of CSF-1 in the primary sensory neurons of the dorsal root ganglion contributes importantly to microglial activation and pro-nociceptive gene induction in animals with neuropathic pain (Guan et al., 2016, Okubo et al., 2016). It is unknown whether CSF-1 plays any role in other types of chronic pain conditions. Furthermore, downstream signaling pathways regulated by CSF-1 in the spinal cord remain not well understood.

In this study, we first characterized the development of chronic pain in the lupus prone mouse model. We found that spinal microglia and astrocytes were activated in MRL/lpr mice with chronic pain, and demonstrated that abnormal functions of CSF-1, IL-1β, and glial glutamate transporters in the spinal dorsal horn are critically implicated in the genesis of chronic pain in MRL/lpr mice.

Material and Methods

Animals

Adult female MRL/MpJ-faslpr (MRL/lpr) and MRL/MpJ (MRL control) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Animals were housed 4 or 5 per cage in isolated rooms with a 12 hour light cycle (8:00 to 20:00). All experiments were approved by the Institutional Animal Care and Use Committee at the University of Georgia and were fully compliant with the National Institutes of Health Guidelines for the Use and Care of Laboratory Animals.

Behavior tests

The mice were placed on a glass surface at 30°C under a Plexiglass cage (12 × 20 × 15 cm), and allowed to acclimate 1 hour and 30 minutes. To measure the thermal sensitivity in the animals, a radiant heat beam was pointed to the mid-plantar surface of each hind paw to evoke a withdrawal response. The time between the stimulus onset and paw withdrawal responses, i.e., the paw withdrawal response latency was measured (Hargreaves et al., 1988). A cutoff time of 20 seconds was used to avoid damage to the skin. Each hind paw was stimulated 3 times and an interval of at least 2 minutes was allowed between the stimulations. The 3 latencies obtained from each paw were averaged. To determine mechanical sensitivity in the animals, mice were placed on a wire mesh, loosely restrained under the Plexiglass cage. von Frey monofilaments with bending force ranging from 0.07 to 2.00 g were used to evoke hind paw withdrawal responses. Each von Frey filament was applied 5 times to the mid-plantar surface of each hind paw for about 1 s. The percentage of withdrawal responses [(number of withdrawal responses of both hind paws/10) × 100%] for each von Frey filament was calculated. Withdrawal response mechanical threshold was defined as the minimum force that evoked greater than 50% responses (Nie et al., 2010). The experimenter conducting the behavioral tests was blind to types of mice and the treatments given to the mice.

Intrathecal drug administration

The CSF-1 receptor inhibitor (GW2580), IL-1 receptor inhibitor (AF12198) was injected into the spinal lumbar enlargement through lumbar puncture (Hylden and Wilcox, 1980, Xu et al., 2006). Briefly, drugs in a volume of 10 μL were intrathecally injected through the L5-6 lumbar interspace in animals anesthetized with 2% isoflurane using a 0.5-inch 30-gauge needle connected to a Hamilton syringe. This was followed by 5 μL of saline injection for flushing. Vehicle was also injected in the same fashion in control groups.

Western blot experiments

The L4 to L5 spinal segment was exposed and removed from the animals deeply anesthetized with urethane (1.3–1.5 g/kg, i.p.). The dorsal half of the spinal cord at the L4 to L5 segment was isolated and quickly frozen in liquid nitrogen and stored at −80°C for later use. The animals were then euthanized. The frozen tissues were homogenized in lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% deoxycholic acid, 2 mM orthovanadate, 100 mM NaF, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 20 μM leupeptin, 100 IU mL−1 aprotinin, pH = 7.5) and placed for 0.5 hr at 37°C. The samples were then centrifuged for 20 min at 12,000 g at 4°C and the supernatants were obtained. Protein concentrations were measured using the Pierce BCA method (Thermo Scientific). Protein samples were loaded onto 10% SDS polyacrylamide gels and electrophoresed. The protein samples were then transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were blocked with 5% milk and incubated overnight at 4°C with the following antibodies: a mouse anti-IL-1β (1:1000, Millipore), anti-GFAP (1:2000, Cell Signaling), anti-OX-42 (1:1000, Cell Signaling), anti-GLT-1 (1:1000, Cell Signaling), anti-CSF-1 (1:1000, ThermoFisher Scientific) antibody, or a monoclonal mouse anti-β-Actin (1:2000, Sigma-Aldrich, St. Louis, USA) primary antibody as a loading control. The blots were then incubated at room temperature for 1 hr with the corresponding HRP-conjugated secondary antibody (1:5000; Santa Cruz Biotechnology, CA, USA), visualized in ECL solution (SuperSignal West Chemiluminescent Substrate, Pierce, Rockford, IL, USA), and exposed on the FluorChem HD2 System. The intensity of immunoreactive bands was analyzed using ImageJ 1.46 software (NIH). Results are expressed as the ratio of each protein over β-Actin control.

Recording and analysis of glial glutamate transporter currents (GTCs) from astrocytes

The spinal lumbar enlargement segment was removed from animals deeply anesthetized via isoflurane inhalation. (Weng et al., 2007, Nie and Weng, 2009). The lumbar spinal cord section was then placed in ice-cold sucrose artificial cerebrospinal fluid (aCSF) saturated with 95% O2 and 5% CO2. The sucrose aCSF contained 234 mM sucrose, 3.6 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 1.2 mM NaH2PO4, 12.0 mM glucose, and 25.0 mM NaHCO3. The L4-5 spinal section was glued to a cutting support with cyanoacrylate, which was then fixed on the stage of a vibratome (Series 1000, Technical Products International, St. Louis, MO). Transverse spinal cord slices (350 μm thick) were cut in the ice-cold sucrose aCSF and then pre-incubated in Krebs solution oxygenated with 95% O2 and 5% CO2 at 35°C. The Krebs solution contained: 117.0 mM NaCl, 3.6 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 1.2 mM NaH2PO4, 11.0 mM glucose, and 25.0 mM NaHCO3 at 35°C. Astrocytes in the superficial spinal dorsal horn were first stained with the astrocyte specific dye, sulforhodomine 101 (100 μM) (Chen et al., 2012), which was pressure-injected into the spinal slice through a pipette with a picospritzer as described previously (Yan et al., 2014, Yan et al., 2015). The labeled astrocyte was patched using a borosilicate glass recording electrode (resistance, 4–6 MΩ) filled with (in mM) 145 potassium-gluconate, 5 NaCl, 1 MgCl2, 0.2 EGTA, 10 HEPES, 2 Mg-ATP and 0.1 Na-GTP (pH 7.3, 290–300 mOsm) (Yan et al., 2015). The membrane potential was hold at −80 mV and the bath perfusion solution contained blockers of GABAA receptor (10 μM bicuculline), glycine receptor (5 μM strychnine), AMPA/kainate receptors (10 μM DNQX), NMDA receptor (25 μM D-AP5), and tetrodotoxin (1 μM) at 35°C. GTCs were evoked by L-glutamate (50 μM) which was injected onto the recorded astrocyte through a glass pipette connected to a Picospritzer controlled by a computer (Yan et al., 2014, Yan et al., 2015). GTCs were recorded before, during drug perfusion, and after washout of drug perfusion. Tested drugs were applied into the recording bath and perfused for 10 min. An average of four GTCs was used for analysis.

Materials

Recombinant CSF-1 (mouse) was obtained from Sigma-Aldrich (Saint Louis, MO). GW2580 (5-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)-pyrimidine-2,4-diamine) was purchased from Calbiochem (San Diego, CA). IL-1, IL-1ra and AF12198 were purchased from R&D System (Minneapolis, MN).

Statistical Analysis

Data are presented as the mean ± standard error (S.E). One or two way analysis of variance (ANOVA) with repeated measurements was used to determine differences on nociceptive behaviors between baseline and each following time point in the same group (One-way ANOVA), or between mice receiving different treatments (two-way ANOVA), This was followed by a Bonferroni post-hoc test to determine sources of the differences. Whenever applicable, Student’s t-tests were used to compare differences between groups (non-paired) or within the same group (paired). Comparisons were run as two-tailed tests. A p value less than 0.05 was considered statistically significant.

Results

MRL/lpr mice develop thermal hyperalgesia and mechanical allodynia in the hind paw

To determine whether chronic pain develops in lupus animal models, withdrawal response thresholds of both hind paws for mechanical stimuli and latencies for heat stimuli were measured in MRL/lpr mice (n=18) and MRL control mice (n=12) weekly from the age of 10 weeks. We found that the withdrawal latencies to radiant heat stimuli in MRL/lpr mice during the 10 to 12 week period were stable and similar to those in MRL control mice. The withdrawal latencies at week 13 of age were significantly shorter in MRL/lpr mice in comparison with their own values at the age of 10 weeks and those obtained from MRL control mice (Fig. 1A). This difference became larger with age, so that the latency of withdrawal responses was significantly reduced by 18.56 ± 3.83% (n = 18, p < 0.001) at the age of 15 weeks and by 19.11± 2.14% (n = 18, p < 0.001) at the age of 16 weeks. The mechanical thresholds of MRL/lpr mice tended to be lower at the age of 14 weeks. This difference became more pronounced with age but it did not reach statistical significance until the mice reached age of 16 weeks (Fig. 1B). MRL/lpr mice within this observation period did not display changes in their motor functions, which included grooming, postures, and gaits. In contrast, mechanical and thermal thresholds of hind paw withdrawal responses in MRL control mice remained stable during the 10 to 16 week observation period (Fig. 1, A and B). Thus, data presented in the rest of the result section below were all from MRL control mice and MRL/lpr mice with chronic pain at the age of 16 weeks.

Figure 1. MRL/lpr mice develop thermal hyperalgesia and mechanical allodynia in the hind paw.

Figure 1

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Line plots show summaries of the withdrawal response latency to heat stimuli (A) and withdrawal response threshold to mechanical stimuli (B) between the ages of 10 weeks to 16 weeks in MRL/lpr mice (n = 16) and MRL control mice (n = 12). Comparisons between MRL/lpr mice and MRL control mice are labeled with +. Comparisons between data at 10 weeks and at each following week are indicated with #. Two symbols: p < 0.01; Three symbols: p < 0.001.

Microglia and astrocytes in the spinal dorsal horn are activated in MRL/lpr mice, which are accompanied with increased protein expression of CSF-1 and IL-1β

Similar to patients with SLE, MRL/lpr mice reportedly display a wide range of pathologic changes like arthritis (Yoneda et al., 2004, Cox et al., 2010), myositis (Edwards et al., 1986), vasculitis (Yamada et al., 2003). Any of these pathological changes could be a cause of chronic pain in MRL/lpr mice. In this study, we chose to focus on the spinal mechanisms implicated in the genesis of chronic pain in MRL/lpr mice. This would allow us to examine the convergent effects induced by pathological changes in different peripheral sites on the common nociceptive sensory processing center (spinal dorsal horn). To investigate whether the activation of glial cells is involved in the development of chronic pain in MRL-lpr mice, we measured protein expressions of the microglial marker (OX-42) and astrocytic marker (GFAP) in the spinal dorsal horn using western blot. As shown in Figure 2, protein expressions of both OX-42 and GFAP were significantly increased in MRL/lpr mice in comparison with MRL control mice, indicating that both microglia and astrocytes were activated in MR/lpr mice with chronic pain. Previous studies showed that microglial activation and proliferation are regulated by CSF-1 (Chu et al., 2016, Guan et al., 2016, Okubo et al., 2016) and IL-1β is an important inflammatory cytokine released from activated microglia and astrocytes in the spinal dorsal horn. Thus, protein expression levels of CSF-1 and IL-1β in the spinal dorsal horn were analyzed using western blot. We found that protein expression levels of CSF-1 and IL-1β in MR/lpr mice were significantly higher than those in MRL control mice. These data suggest that CSF-1 and IL-1β may be implicated in the spinal synaptic plasticity in MRL/lpr mice with chronic pain.

Figure 2. MRL/lpr mice with chronic pain have activation of microglia and astrocytes, and increased protein expressions of CSF-1 and IL-1β in the spinal dorsal horn.

Figure 2

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(A): Samples of protein expressions of OX-42, GFAP, CSF-1, and IL-1β in the spinal dorsal horn of MRL/lpr mice (n = 5) and MRL control mice (n = 5) are shown. (B): Bar graphs show the mean (+SE) density of each molecule relative to β-actin in both groups. *** p < 0.001.

Blockade of CSF-1 receptors or IL-1β receptors ameliorates thermal hyperalgesia in MRL/lpr mice

To determine if CSF-1 in the spinal cord plays a role in the genesis of chronic pain in MRL/lpr mice, the effects of the CSF-1 receptor specific inhibitor GW2580 on thermal hyperalgesia in MRL/lpr mice were measured. Animals at 16 weeks old were randomly assigned into 4 groups: MRL/lpr mice + GW2580 group (n = 6); MRL/lpr mice + vehicle group (n = 5); MRL control mice + GW2580 group (n = 6); and MRL control mice + vehicle group (n = 6). For the MRL/lpr mice GW2580 group and MRL control mice + GW2580 group, GW2580 (concentration: 100 μM in a volume of 10 μL) was intrathecally injected via lumbar puncture. Vehicle at the same volume was injected into mice in the MRL/lpr mice + vehicle group and MRL/lpr mice + vehicle group in the same fashion. The latencies of hind paw withdrawal responses to radiant heat stimuli before (baseline) and after the intrathecal injection were measured. As shown in Figure 3A, at 30 min after the injection, the latency of withdrawal responses in the MRL/lpr mice + GW2580 group were significantly increased in comparison with their own baseline values or with the MRL/lpr mice + vehicle group. Injection of GW2580 did not significantly alter the latency of withdrawal responses in the MRL control mice + GW2580 group. Meanwhile, the latency of withdrawal responses was not significantly altered in either the MRL control mice + vehicle group or the MRL/lpr mice + vehicle group. These data indicate that activation of CSF-1 receptors in implication in the thermal hypersensitivity in MRL/lpr mice. This notion is further supported by another set of experiments, in which we determined the thermal sensitivity in MRL control mice following CSF-1 treatment. MRL control mice were randomly assigned into two groups to receive intrathecal injection of either recombinant CSF-1 (25 ng, in a volume of 10 μL) or vehicle only. We found that in comparison with its own baseline values (prior to intrathecal injection) or those in MRL control mice (n = 5) treated with vehicles, MRL control mice (n = 6) receiving CSF-1 had significant shorter response latencies to radiant heat stimuli (Fig. 3B).

Figure 3. Blockade of CSF-1 receptors or IL-1β receptors ameliorates thermal hyperalgesia in MRL/lpr mice.

Figure 3

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(A): Line plots show measurements of the hind paw withdrawal response latency in MRL/lpr mice and MRL control mice to heat stimuli collected at baseline (prior to treatments), and then 30, 60, 90, 120, and 180 minutes after the intrathecal administration of either GW2580 (concentration:100 μM, 10 μL) or vehicle (10 μL). Number of animals used per group: MLR/lpr + vehicle, n = 5; MRL control + vehicle, n = 6; MRL/lpr + GW2580 (100 μM), n = 6; MRL control + GW2580 (100 μM), n = 6. (B): Line plots show measurements of the hind paw withdrawal response latency in MRL control mice to heat stimuli collected at baseline, and then 30, 60, 90, 120, and 180 min after the intrathecal administration of either CSF-1 (25 ng) (n = 6) or vehicle only (n = 5). For both (A) and (B), comparisons between the drug-treated group and vehicle-treated group are labeled with +. Comparisons between data at baseline and at each following time point within the same drug-treated group are indicated with #. (C): Line plots show measurements of the hind paw withdrawal response latency in MRL/lpr mice and MRL control mice to heat stimuli collected at baseline, and then 30, 60, 90, 120, and 180 minutes after the intrathecal administration of either AF12198 (concentration:100 μM or 400 μM, 10 μL) or vehicle (10 μL) only. Number of animals used per group: MLR/lpr + vehicle, n = 5; MRL control + vehicle, n = 5; MRL/lpr + AF12198 (100 μM), n = 6; MRL control + AF12198 (100 μM), n = 6; MRL/lpr + AF12198 (400 μM), n = 6; MRL control + AF12198 (400 μM), n = 6. Comparisons between the AF12198 (400 μM)-treated MRL/lpr group and vehicle-treated MRL/lpr group are labeled with +. Comparisons between data at baseline and at each following time point within the AF12198 (400 μM)-treated MRL/lpr group are indicated with #. Comparisons between the AF12198 (100 μM)-treated MRL/lpr group and vehicle-treated MRL/lpr group are labeled with ^. Comparisons between data at baseline and at each following time point within the AF12198 (100 μM)-treated MRL/lpr group are indicated with *. Two symbols: p < 0.01; Three symbols: p < 0.001.

Next, we defined the role of IL-1β in the genesis of chronic pain in MRL/lpr mice. Two different doses of the IL-1 receptor specific blocker AF12198 (Akeson et al., 1996) (either a concentration of 100 μM or 400 μM at 10 μL) were respectively given to two groups of MRL/lpr mice via lumbar puncture. Two MRL control groups were treated with AF12198 in the same manner. Vehicle in a volume of 10 μL were intrathecally injected into another MRL/lpr group and MRL control group. In MRL/lpr mice with chronic pain, AF12198 prolonged the latency of withdrawal responses to radiant heat stimuli in a dose-dependent manner in comparison with its own baseline values or with those in MRL/lpr mice treated with vehicle (Fig. 3C). Latencies of withdrawal responses to heat stimuli in MRL control mice treated with AF12198 or vehicle were not significantly altered. Our findings suggest that IL-1β contributes importantly to thermal hyperalgesia in MRL/lpr mice, consistent with previous reports that intrathecal injection of IL-1β induces thermal hyperalgesia (Sung et al., 2004).

Protein expression and activities of glial glutamate transporters in the spinal dorsal horn of MRL/lpr mice with chronic pain are reduced

It has been previously demonstrated that dysfunction of glial glutamate transporters plays a critical role in the excessive activation of glutamate receptors of neurons in the spinal dorsal horn in animals with neuropathic pain (Nie and Weng, 2009, 2010). To determine protein expression of glial glutamate transporters, we compared glial glutamate transporter 1 (GLT-1) protein expression in the spinal dorsal horn of MRL control and MRL/lpr mice with chronic pain using western blot. As shown in Figure 4A, protein expression of GLT-1 was lower in MRL/lpr mice (N = 5) than MRL control mice (n = 5). We further compared glial glutamate transporter activities in MRL/lpr mice and MRL control mice by recording glutamate transporter currents (GTCs) from astrocytes in the spinal superficial laminae. Glutamate uptake by glial glutamate transporters is accompanied by the co-transport of two or three Na+ with one H+ and the counter-transport of one K+ (Wadiche et al., 1995, Levy et al., 1998, Tegeder et al., 2008), which results in a generation of currents termed GTC (Wadiche et al., 1995, Levy et al., 1998, Tegeder et al., 2008). By recording GTCs, the function of glial glutamate transporters can be directly monitored in real time. We identified astrocytes by labeling astrocytes with the astrocyte specific dye, sulforhodomine 101 (100 μM) (Chen et al., 2012). The accuracy of such techniques has been previously confirmed by us using single-cell RT-PCR (Yan et al., 2015). GTCs were evoked by L-glutamate (50 μM) puffed onto the astrocyte through a puff-electrode (Yan et al., 2015). We found that the GTC amplitude and charge transfer (area of GTC) were significantly lower in MRL/lpr mice (45 astrocytes) than MRL control mice (54 astrocytes) (Fig. 4B). These data demonstrate that the glial glutamate transporter function in MRL/lpr mice was impaired.

Figure 4. Protein expression and activities of glial glutamate transporters in the spinal dorsal horn of MRL/lpr mice with chronic pain are reduced.

Figure 4

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(A): Samples of protein expressions of GLT-1 in the spinal dorsal horn of MRL/lpr mice and MRL control mice are shown. (B): Bar graphs show the mean (+SE) density of GLT-1 relative to β-actin in both groups. (C): Samples of GTCs recorded from astrocytes in the spinal dorsal horn of MRL/lpr mice and MRL control mice are shown. Bar graphs show the mean (+SE) amplitude and charge transfer of GTCs in MRL/lpr mice and MRL control mice. *** p < 0.001.

Blockade of CSF-1 receptors or IL-1β receptors enhances glutamate transporter activities in the spinal dorsal horn in MRL/lpr mice

To investigate spinal mechanisms by which CSF-1 regulates chronic pain in MRL/lpr mice, we determined whether glial glutamate transporter activities are under the regulation of CSF-1. GTCs were recorded from astrocytes of the spinal dorsal horn in the spinal slices. GTCs recorded before, during and after washout of drug perfusion were analyzed. In GTCs recorded from spinal slices of MRL/lpr mice, we found that bath-perfusion of the CSF-1 inhibitor (GW2580; bath concentration: 10 μM) significantly increased peak amplitudes and charge transfers of GTCs, and these effects went away after washout of GW2580 (Fig. 5A). Furthermore, when we recorded GTCs from spinal slices of MRL control mice, bath-perfusion of recombinant CSF-1 (bath concentration: 25 ng/mL) significantly reduced peak amplitudes and charge transfers of GTCs (Fig. 5B). These data indicate that CSF-1 causes spinal central sensitization at least in part via decreasing glial glutamate transporter activities.

Figure 5. Blockade of CSF-1 receptors enhances glutamate transporter activities in the spinal dorsal horn in MRL/lpr mice.

Figure 5

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(A): Shown are samples of GTCs recorded from astrocytes in the spinal dorsal horn of MRL/lpr mice before (baseline), during and after washout of GW2580 (bath concentration:10 μM) perfusion. (B): Bar graphs show the mean (+SE) amplitude and charge transfer of GTCs in MRL/lpr mice at baseline, during and after washout of GW2580. (C): Shown are samples of GTCs recorded from astrocytes in the spinal dorsal horn of MRL control mice before, during and after washout of CSF-1 (bath concentration: 25 ng/mL) perfusion. (D): Bar graphs show the mean (+SE) amplitude and charge transfer of GTCs in MRL/lpr mice before, during and after washout of CSF-1 perfusion. The number of astrocytes included in each group for the analysis is shown in each bar. * p < 0.05; ** p < 0.01; *** p < 0.001.

We then determined whether spinal glial glutamate transporter activities in MRL/lpr mice are under the control of IL-1β. We used the IL-1 antagonist IL-1ra to block the effect of IL-1β, which is widely used in electrophysiological experiments. We found that bath-perfusion of IL-1ra (bath concentration: 10 μM) significantly enhanced peak amplitudes and charge transfers of GTCs recorded from MRL/lpr mice (Fig. 6A). Furthermore, GTCs recorded from MRL control mice were attenuated by bath-perfusion of IL-1β (bath concentration: 10 ng/mL) (Fig. 6B). These data indicate that IL-1β is implicated in the generation of thermal hyperalgesia through suppressing spinal glial glutamate transporter activities.

Figure 6. Blockade of IL-1β receptors enhances glutamate transporter activities in the spinal dorsal horn in MRL/lpr mice.

Figure 6

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(A): Shown are samples of GTCs recorded from astrocytes in the spinal dorsal horn of MRL/lpr mice before (baseline), during and after washout of IL-1ra (bath concentration: 10 μM) perfusion. (B): Bar graphs show the mean (+SE) amplitude and charge transfer of GTCs in MRL/lpr mice at baseline, during and after washout of IL-1ra. (C): Shown are samples of GTCs recorded from astrocytes in the spinal dorsal horn of MRL control mice before, during and after washout of IL-1β (bath concentration: 10 ng/mL) perfusion. (D): Bar graphs show the mean (+SE) amplitude and charge transfer of GTCs in MRL control mice before, during and after washout of IL-1β perfusion. The number of astrocytes included in each group for the analysis is shown in each bar. * p < 0.05; ** p < 0.01; *** p < 0.001.

IL-1β mediates the effects of CFS-1 on glial glutamate transporters

Given that CSF1 receptors are only expressed in microglia in the spinal dorsal horn (Guan et al., 2016) and activation of microglia results in release of inflammatory cytokines such as IL-1β from microglia, we speculated that IL-1β mediates the effects of CFS-1 on glial glutamate transporters. We tested this speculation by recording GTCs from spinal slices obtained from MRL control mice. After recording the baseline GTCs, recombinant CSF-1 (bath concentration: 25 ng/mL) was perfused into the recording bath. We reconfirmed that CSF-1 significantly suppresses the amplitude and charge transfer of GTCs (n = 10; p < 0.001) (Fig. 7A). This suppression was reversed when the IL-1 receptor blocker IL-1ra (10 μM) was added into the recording chamber in the presence of CSF1 (n = 10; p < 0.001) (Fig. 7a). In another set of experiments, GTCs were recorded from spinal slices of MRL/lpr mice. After confirming that bath-perfusion of the CSF-1 inhibitor (GW2580; bath concentration: 10 μM) significantly increased peak amplitudes and charge transfers of GTCs (n = 15; p < 0.001) (Fig. 7B), we added IL-1ra (10 μM) into the recording chamber in the presence of GW2580. Further addition of IL-1ra did not significantly alter the amplitude and charge transfer of GTCs (Fig. 7B). These data highly suggest that IL-1β is a downstream signaling molecule mediating the effects of CFS-1 on glial glutamate transporters.

Figure 7. IL-1β mediates the effects of CFS-1 on glial glutamate transporters.

Figure 7

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(A): Shown are samples of GTCs recorded from astrocytes in the spinal dorsal horn of MRL control mice before and during perfusion of CSF-1 (25 ng/mL), as well as further addition of IL-1ra (bath concentration: 10 μM) in the presence of CSF-1. (B): Bar graphs show the mean (+SE) amplitude and charge transfer of GTCs in MRL control mice before and during perfusion of CSF-1, as well as further addition of IL-1ra in the presence of CSF-1. (C): Shown are samples of GTCs recorded from astrocytes in the spinal dorsal horn of MRL/lpr mice before (baseline) and during perfusion of GW2580 (bath concentration:10 μM), as well as further addition of IL-1ra (bath concentration: 10 μM) in the presence of GW2580. (B): Bar graphs show the mean (+SE) amplitude and charge transfer of GTCs in MRL/lpr mice before and during perfusion of GW2580, as well as further addition of IL-1ra in the presence of GW2580.

Discussion

Our current study provides the first characterization of spinal mechanisms underlying chronic pain in MRL/lpr mice. We found that female MRL/lpr mice developed thermal hyperalgesia at the age of 13 weeks, and mechanical allodynia at the age of 16 weeks. MRL/lpr mice with chronic pain have activation of microglia and astrocytes, over-expression of CSF-1 and IL-1β, as well as suppression of glial glutamate transport function in the spinal cord. CSF-1 and IL-1β regulates the spinal pain signaling via suppressing glial glutamate transporter activities. Our findings suggest that spinal neuroinflammation and enhanced glutamatergic synaptic activities underlie the spinal molecular and synaptic mechanisms in MRL/lpr mice with chronic pain.

Targeting glial glutamate transporters for the treatment of chronic pain

Aberrant neuronal activation in the spinal dorsal horn plays an important role in pathological pain conditions induced by nerve injury (Yan and Weng, 2013), inflammation in peripheral tissues (Liu et al., 2008, Ren and Dubner, 2010, Ji et al., 2013), or chemotherapy-agents (Weng et al., 2003, Cata et al., 2006). Aberrant neuronal activities in the spinal dorsal horn can be caused by changes in the active and passive electrophysiological membrane properties of the neurons, in the balance between presynaptic excitatory and inhibitory input to neurons, and in the functions of postsynaptic excitatory and inhibitory ligand-gated ion channels. Over-activation of AMPA receptors and NMDA glutamate receptors in the spinal dorsal horn plays a critical role in abnormal neuronal activation in pathological pain conditions (Yan and Weng, 2013). While administration of glutamate receptor blockers (like AMPA or NMDA antagonists) reduces nociceptive behavioral responses in animals (Lufty et al., 1997, Nishiyama et al., 1998, Zahn et al., 1998) and pathologic pain in patients (Kristensen et al., 1992, Wu and Zhuo, 2009), glutamate receptor blockers produce intolerable side effects like motor impairment and psychotomimetic adverse effects at analgesic doses (Lufty et al., 1994, Wu and Zhuo, 2009). Studies by others and us have shown that activation of glutamate receptors in the spinal dorsal horn is controlled by glial cells, thus one may normalize activation of glutamate receptors by regulating glial functions and therefore avoid the side-effects caused by direct inhibition of glutamate receptors. One key mechanism by which glia regulates neuronal activation is that astrocytes maintain glutamate homeostasis by up-taking glutamate via glial glutamate transporters since glutamate released from neurons cannot be metabolized extracellularly (Danbolt, 2001). We and others have demonstrated that downregulation of protein expression of glial glutamate transporters in the spinal dorsal horn is associated with neuropathic pain induced by nerve injury (Nie et al., 2010) or chemotherapy (paclitaxel) (Weng et al., 2005), inflammatory pain induced by CFA (Kim et al., 2012), and morphine tolerance (Mao et al., 2002). Deficient glutamate uptake by glial cells is key to enhanced activation of glutamate receptors in spinal nociceptive neurons and the genesis of neuropathic pain induced by peripheral nerve injury (Weng et al., 2007, Nie and Weng, 2010). Enhanced glial glutamate transporter expression via treatments of ceftriaxone (Hu et al., 2010, Ramos et al., 2010), gene transfer (Maeda et al., 2008) or glial modulation with propentofylline (Tawfik et al., 2008) or minocycline (Nie et al., 2010) significantly reduces allodynia induced by nerve injury in rodents. Currently, the role of glial glutamate transporters in the generation of chronic pain in SLE is unknown. In this study, we for the first time demonstrated that impaired glial glutamate transport in the spinal dorsal horn is implicated in the genesis of chronic pain in MRL/lpr mice. We previously reported that the functional integrity of glial glutamate transporters in the spinal cord is required not only for the normal activities of AMPA and NMDA receptors21,30, but also for the glutamate-glutamine cycle-dependent GABA synthesis (Jiang et al., 2012). Thus, it is conceivable that both the enhancement of glutamatergic synaptic activities and suppression of GABAergic synaptic activities contribute to over-activation of spinal dorsal horn neurons in MRL/lpr mice with chronic pain. Given the critical role of glial glutamate transporters in the generation of different chronic pain conditions, identifying molecular targets that can improve the function of glial glutamate transporters is a promising avenue for the development of analgesics.

The role of M-CSF1 in the pain signaling system

Previous studies have shown that CSF-1 is essential to maintaining microglial population. Mice with CSF-1 mutation have a reduced population of microglia and impaired microglial activation (Berezovskaya et al., 1995, Kalla et al., 2001). Our understanding of the role of CSF-1 in the pain signaling system is limited. It was shown that intra-articular injection of CSF-1 antibody attenuates pain in animals with arthritis induced by injection of CFA into the knee joint (Alvarado-Vazquez et al., 2015). Protein expression of CSF-1 is up-regulated in the spinal dorsal horn of rats that develop allodynia following a brief electric stimulation of peripheral C-fibers (Hathway et al., 2009). More recently, CSF-1 was found to be a key molecule mediating communication between the injured primary sensory neurons and microglia in the spinal dorsal horn of animals with neuropathic pain (Guan et al., 2016, Okubo et al., 2016). Peripheral nerve injury significantly increases CSF-1 protein expression in the primary sensory neurons (Guan et al., 2016, Okubo et al., 2016). In the spinal dorsal horn CSF-1 receptor is only expressed in microglia (Guan et al., 2016). CSF-1 contributes to the generation of neuropathic pain through stimulation of microglial proliferation and induction of microglial genes encoding cathepsin S and brain-derived neurotrophic factor (Guan et al., 2016). In the present study, we for the first time demonstrated that CSF-1 plays a critical role in the genesis of chronic pain in MRL/lpr mice. The concurrently increased expression of CSF-1 with activation of microglia and increased expression of IL-1β (Fig. 2) in MRL/lpr mice with chronic pain is in agreement with the role of CSF-1 in the regulation of microglial activation and proliferation reported previously. Intriguingly, we found that thermal hyperalgesia in in MRL/lpr mice is attenuated within 30 min after intrathecal injection of the CSF-1 receptor blocker while glial glutamate transporter activities recorded from spinal slices of MRL/lpr mice are increased within 10 min of bath-perfusion of the CSF-1 receptor blocker. These data indicate that the ongoing activation of CSF-1 is needed to maintain the hyperalgesia state and suppression of glial glutamate transporter activities in MRL/lpr mice. This notion is further supported by our findings that administration of recombinant CSF-1 into the intrathecal space of MRL control mice induces thermal hyperalgesia within 30 min, and glial glutamate transporter activities recorded from spinal slices of MRL control mice are attenuated within 10 min of bath-perfusion of recombinant CSF-1. The rapid responses to CSF-1 and the CSF-1 receptor blocker are incompatible to the time scale needed for CSF-1 to regulate microglial proliferation and the protein synthesis from gene-induction. Rather, it may suggest an engagement of post-translational mechanisms. Indeed, it has been demonstrated that prior to causing cell-proliferation and gene-induction, activation of CSF-1 receptors in microglia can rapidly increase activities of NADPH oxidase in microglia (Imai and Kohsaka, 2002). Activation of NADPH oxidase in microglia results in secretion of IL-1β via activation of NLRP3 inflammasome (Bordt and Polster, 2014). Based on our findings that IL-1β mediates the suppressive effect induced by CSF-1 on glial glutamate transporter activities (Fig. 7), it is likely that the same signaling pathway is also used by CSF-1 in the spinal dorsal horn. Further studies are warranted to confirm such speculation.

The role of IL-1β in the generation of pathological pain

IL-1β is a prototypic pro-inflammatory cytokine, secreted from activated microglia and astrocytes in the spinal dorsal horn (Beggs and Salter, 2016, Ren and Dubner, 2016). Increased production of IL-1β in the spinal dorsal horn has been reported in many animal models with pathological pain like those induced by nerve injury (Yan and Weng, 2013), paclitaxel-chemotherapy (Gao et al., 2013), bone cancer (Zhang et al., 2008b), inflammation caused by CFA (Zhang et al., 2008a). Here, we extended the role of IL-1β to the genesis of chronic pain in lupus prone mice, as evident by an increased production of IL-1β in MRL/lpr mice and attenuation of thermal hyperalgesia by the IL-1β antagonist. It has been reported that IL-1β enhances the frequency and amplitude of spontaneous EPSCs and currents induced by AMPA and NMDA in dorsal horn neurons, indicating that both the release of glutamate from presynaptic neurons and functions of AMPA and NMDA receptors in postsynaptic neurons are increase by IL-1β (Yan and Weng, 2013). Furthermore, we demonstrated that myeloid differentiation primary response protein 88 (MyD88) is required for IL-1β to enhance postsynaptic non-NMDA glutamate receptor activities in the spinal dorsal horn, and presynaptic NMDA receptors are effector receptors for IL-1β to enhance glutamate release from the primary afferents in animals with neuropathic pain (Yan and Weng, 2013). The neutral sphingomyelinase/ceramide signaling pathway mediates the functional coupling between IL-1β receptors and presynaptic NMDA receptors at the primary afferent terminals (Yan and Weng, 2013). In this study, we revealed that endogenous IL-1β in the spinal dorsal horn of MRL/lpr mice with chronic pain causes suppression of glial glutamate transporter activities. The similar effects were reported in rats with neuropathic pain (Yan and Weng, 2013). These findings re-emphasize the crucial role of IL-1β and glial glutamate transporters in the genesis of pathological pain conditions.

The role of microglia in the genesis of pathological pain in different genders

The role of microglia in the genesis of pathological pain in different genders remains controversial. Some reports suggest that activation of microglia and toll like receptors 4 (TLR4), which are predominantly expressed in microglia, are important in the genesis of mechanical allodynia only in male but not female animals. For example, intrathecal injection of the microglia inhibitor minocycline reverses mechanical allodynia induced by spared nerve injury or persistent inflammatory pain induced by CFA in male mice but not in female mice (Sorge et al., 2015). Activation of spinal TLR4 with intrathecal injection of lipopolysaccharide induces mechanical allodynia in male but not in female mice (Sorge et al., 2011). However, evidence supporting the critical role of microglia in the genesis of pathological pain in female animals is also reported. Depletion of microglia and macrophages in the CNS and dorsal root ganglion abolishes the development of mechanical and thermal hypersensitivity induced by spinal nerve transection in both male and female mice (Peng et al., 2016). Gene-knockdown of TLR4 prevents and reverses bone cancer pain in female rats (Liu et al., 2013). Blocking the action of spinal endogenous TLR4 ligand (Agalave et al., 2014) or suppressing microglial activation (Nieto et al., 2016) reverses mechanical hypersensitivity in female mice with collagen antibody-induced arthritis. Our study provides the evidence that activation of microglial CSF-1 receptors and IL-1 receptors is critical to the maintenance of thermal hyperalgesia in female lupus mice. Importantly, by using the electrophysiological approach, we show that activities of spinal glial glutamate transporters in female mice are reversibly suppressed by activation of CSF1 receptors or IL-1β receptors. Taking together all the findings by others and us, it is conceivable that the role of microglia in the genesis of pathological pain in different genders may be etiology-specific and sensory modality-specific.

Conclusion

Our study for the first time characterized the development of chronic pain in the lupus prone animal model. Our findings suggest that targeting the CSF-1 signaling pathway or the glial glutamate transporter in the spinal cord is an effective approach for the management of chronic pain caused by SLE.

Acknowledgments

This project was supported by the NIH RO1 grant (NS064289) to H.R.W. and in part by Hubei Province Natural Science Fund for Young Scholars (NO. 2016CFB120), Hubei Province Health and Family Planning Scientific Research Project (NO. WJ2017Q033), Wuhan Youth Science and Technology Plan (NO. 2015071704011627), Wuhan Municipal Population and Family Planning Commission Foundation (NO. WX16A04), and the Fourth Batch of Wuhan Medical Backbone of Middle-aged and Young Talents Plan to X.Y. The authors declare no conflict of interest.

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