Gabapentin reduces CX3CL1 signaling and blocks spinal microglial activation in monoarthritic rats

Abstract

Background: Spinal glia, particularly microglia and astrocytes, are of the utmost importance in the development and maintenance of chronic pain. A recent study from our laboratory revealed that gabapentin, a recommended first-line treatment for multiple neuropathic conditions, could also efficiently antagonize thermal hyperalgesia evoked by complete Freund's adjuvant (CFA)-induced monoarthritis (MA). In the present study, we investigated whether the spinal glia are involved in the anti-hyperalgesic effect of gabapentin and how this event occurs.

Results: Unilateral intra-articular injection of CFA produced a robust activation of microglia and astrocytes. These cells exhibited large cell bodies, thick processes and increases in the ionized calcium binding adapter molecule 1 (Iba-1, a microglial marker) or the glia fibrillary acidic protein (GFAP, an astrocytic marker). These cells also displayed immunoreactive signals, and an upregulation of the voltage-gated calcium channels (VGCCs) α2/δ-1 subunit, CX3CL1 and CX3CR1 expression levels in the spinal cord. These changes were associated with the development of thermal hyperalgesia. Immunofluorescence staining showed that VGCC α2/δ-1 subunit, a proposed gabapentin target of action, was widely distributed in primary afferent fibers terminals and dorsal horn neurons. CX3CL1, a potential trigger to activate microglia, colocalized with VGCC α2/δ-1 subunits in the spinal dorsal horn. However, its receptor CX3CR1 was mainly expressed in the spinal microglia. Multiple intraperitoneal (i.p.) gabapentin injections (100 mg/kg, once daily for 4 days with the first injection 60 min before intra-articular CFA) suppressed the activation of spinal microglia, downregulated spinal VGCC α2/δ-1 subunits decreased CX3CL1 levels and blocked the development of thermal hyperalgesia in MA rats.

Conclusions: Here we provide the first evidence that gabapentin diminishes CX3CL1 signaling and spinal microglia activation induced by joint inflammation. We also show that the VGCC α2/δ-1 subunits might be involved in these events.

Figure 1

The activation of spinal glia (microglia and astrocytes) and thermal hyperalgesia by unilateral intra-articular injection of complete Freund's adjuvant (CFA)-induced monoarthritis (MA). A, Immunohistochemistry reveals an extensive increase in Iba-1- (a microglial marker) immunoreactivity (Iba-1-IR) in the ipsilateral lumbar spinal dorsal horn at 4 hrs and in both sides by days 1 and 4 after MA. B, Immunohistochemistry reveals a marked increase in GFAP- (a microglial marker) immunoreactivity (GFAP-IR) in both sides of the lumbar spinal dorsal horn on day 4 after MA. C and D, Quantification of Iba-1-IR (C) and GFAP-IR (D) expression on both sides of the spinal dorsal horn ** p < 0.01 vs. naïve control; + p < 0.05, ++ p < 0.01 vs. contralateral. E, MA induced significant thermal hyperalgesia in the ipsilateral hindpaw ** p < 0.01 vs. sham MA.

Figure 2

Effects of multiple intraperitoneal (i.p.) injection of gabapentin (Gab, 100 mg/kg) on MA-induced spinal glial activation and thermal hyperalgesia. A and B, Effects of repeated Gab treatment on MA-induced increase in Iba-1-1R (A) and GFAP (B) in the ipsilateral spinal dorsal horn. C and D, Quantification of Iba-1-IR (C) and GFAP-IR (D) expression on both sides of the spinal dorsal horn ** p < 0.01 vs. sham control; + p < 0.05, ++ p < 0.01 vs. MA. E, Repeated Gab treatment inhibited MA-induced thermal hyperalgesia. Repeated i.p. Gab or NS were given directly after the behavioral test on days 0–3, with the first injection 60 min before intra-articular injection of CFA. ** p < 0.01 vs. NS control.

Figure 3

Expression of the voltage-gated calcium channels (VGCCs) α2/δ-1 subunit in the lumbar spinal cord. A, The western blot shows an increase in the level of VGCC α2/δ-1 subunit expression in the ipsilateral lumbar spinal dorsal horn at 4 hrs and 4 days after MA (CFA/NS injected into the ankle articular cavity). B, Western blot analysis reveals that repeated Gab treatment significantly suppresses MA-induced upregulation of the VGCC α2/δ-1 subunit in the ipsilateral lumbar spinal dorsal horn. * p < 0.05, ** p < 0.01 vs. control (sham MA or naïve); ++ p < 0.01 vs. MA. C, Immunohistochemistry reveals that the VGCC α2/δ-1 subunits are extensively distributed in the spinal dorsal horn. Double immunofluorescence staining shows that the VGCC α2/δ-1 subunit does not colocalized with the microglial marker Iba-1 or the astrocytic marker GFAP in the spinal dorsal horn. D, Double immunofluorescence staining shows that VGCC α2/δ-1 subunits are colocalized with IB4 in the inner lamina II and CGRP and SP in the lamina I and outer lamina II of the spinal cord.

Figure 4

Expression of CX3CL1 and its receptor CX3CR1 in the lumbar spinal cord. A, Double immunofluorescence staining shows that CX3CL1 is colocalized with the neuronal marker NeuN and VGCC α2/δ-1 subunits in all of the layers of the spinal dorsal horn. B, The western blot shows an increase in the level of CX3CL1 in the ipsilateral lumbar spinal dorsal horn at 4 hrs and 4 days after MA. C, Western blot analysis reveals that repeated Gab treatment significantly suppresses MA-induced upregulation of CX3CL1 in the lumbar spinal dorsal horn. D, Double immunofluorescence staining shows that CX3CR1 is colocalized with the microglial marker OX-42, but not the astrocytic marker GFAP. E, Western blot analysis reveals that repeated Gab treatment partially inhibits MA-induced upregulation of CX3CR1 in the lumbar spinal dorsal horn. * p < 0.05, ** p < 0.01 vs. control (sham MA or naïve); ++ P < 0.01 vs. MA.

Figure 5

A schematic illustration of spinal glial activation in monoarthritic pain. Joint inflammation may increase the release of nociceptive transmitters and modulators (such as EAAs, SP, ATP and CX3CL1) in the spinal dorsal horn from the primary afferent terminals ipsilateral to the inflammed joint [39]. These events can initiate early focal microglial activation in ipsilateral spinal cord (within 4 hrs after MA). Activated microglia release several proinflammatory cytokines and chemokines that may spread to contralateral spinal cord, leading to the contralateral spinal microglial and astrocytic activation [3]. Once the glia are activated, they release several glial products including proinflammatory cytokines, tumor necrosis factor-α, and inflammatory mediators. This leads to an exaggerated release of neurotransmitters from presynaptic neurons, sensitization of the post-synaptic membrane, activation of neighboring astrocytes, and enhancement of the microglial propagation of neuromediators [5]. Such positive feedback loops sustain the perseverant release of pain mediators, facilitating the development of neuronal hypersensitivity, leading to exaggerated pain (such as thermal hyperalgesia) [13]. Gabapentin might diminish the release of “pain” neurotransmitters/neuromodulators (such as CX3CL1) and activation of microglia in the spinal cord by modulating VGCC α2/δ-1 subunits, leading to a reduction in thermal hyperalgesia.