Induction of the P2X7 Receptor in Spinal Microglia in a Neuropathic Pain Model

Abstract

Peripheral nerve injury causes a progressive series of morphological changes in spinal microglia, and extracellular ATP stimulates proliferation of microglia and may be involved in neuropathic pain. We defined the precise expression of P2X7 in the spinal cord following peripheral nerve injury. We found that both P2X7 mRNA and protein increased in the spinal cord, with a peak at 7 days after injury. Double labeling studies revealed that cells expressing increased P2X7 mRNA and protein after nerve injury were predominantly microglia in the dorsal horn. Pharmacological blockade by intrathecal administration of a P2X7 antagonist (A438079 hydrochloride) suppressed the development of mechanical hypersensitivity. We present distinct evidence that increases in the number of P2X7 receptors in spinal microglia may play an important role in neuropathic pain.

Introduction

Peripheral nerve injury can trigger neurological abnormalities, including neuropathic pain. Recent findings demonstrate the important role of spinal microglial activation following peripheral nerve injury in the pathomechanism of neuropathic pain. Extracellular ATP causes many pathological reactions of microglia, one of which is microglial activation. ATP receptors (P2 receptors) are divided into seven subtypes of ionotropic P2X receptors (P2X1–P2X7) and eight subtypes of metabotropic P2Y receptors. P2X receptors are assembled from different subunits into homo- or heterotrimers and the ATP binding opens the pore permeable to Na⁺, K⁺, and Ca²⁺. It has been shown that the P2X4 receptor in microglia plays crucial roles in neuropathic pain development. Recently, the possible involvement of spinal P2X7 in neuropathic pain has been suggested using pharmacological examinations. In addition, the study with mice with genetic knockout of the P2X7 gene showed a suppression of pain-related behaviors following peripheral nerve injury. Although these studies suggest that P2X7 plays an important role in the pathology of neuropathic pain, the morphological expression of P2X7 in the spinal cord has not been determined. In this study, we examined the precise expression of P2X7 in the spinal cord following peripheral nerve injury, both in terms of mRNA and protein levels, and examined the involvement of P2X7 in neuropathic pain behavior using a specific inhibitor of P2X7.

Materials and Methods

All animal experimental procedures were approved by the Hyogo College of Medicine Committee on Animal Research and were carried out in accordance with the National Institutes of Health guidelines on animal care. Male Sprague Dawley rats weighing 200–250 g were anesthetized with sodium pentobarbital and the tibial and common peroneal nerves were transected, while the sural nerve was left intact (spared nerve injury; SNI model). The wounds were then closed and the rats were allowed to recover. At several time points (1, 3, 7, 14, and 30 days) following the surgery, groups of rats were processed for analysis.

PCR primers for P2X7 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA were designed. Samples for RT-PCR and in situ hybridization (ISHH) were prepared. For ISHH, the L4-5 spinal cord was dissected, frozen, and sectioned for analysis.

For immunohistochemistry (IHC), cryostat sections were fixed, treated with antibodies against Iba1, NeuN, and GFAP, and processed for double labeling with ISHH. The spinal samples were prepared for Western blotting to analyze protein levels of P2X7. The membranes were incubated with antibodies, washed, and visualized by chemiluminescence. Data were analyzed statistically using one-way ANOVA and Fisher’s post hoc tests.

IHC was also performed for fluorescence imaging with antibodies against P2X7, Iba1, NeuN, and GFAP, and visualized using Alexa Fluor secondary antibodies. A preabsorption control experiment was performed to test the specificity of the P2X7 antibody.

For behavioral testing, osmotic pumps were used to deliver A438079 hydrochloride intrathecally after SNI surgery. Rats were tested for mechanical allodynia before and after surgery using a dynamic plantar aesthesiometer.

Results

We examined the expression of P2X7 mRNA in the spinal cord of neuropathic pain model rats. RT-PCR analysis revealed an upregulation of P2X7 mRNA following SNI, with a peak at 7 days and sustained expression until 30 days. ISHH showed that the number of P2X7 mRNA-positive cells increased in the ipsilateral dorsal horn, with stronger signal intensity compared to the contralateral side. Double labeling with ISHH and IHC indicated that P2X7 mRNA was primarily expressed in microglia.

Western blot analysis showed increased P2X7 protein expression in the ipsilateral spinal cord at 7 and 30 days after injury. IHC confirmed that P2X7 protein was upregulated in the dorsal horn microglia. The preabsorption test showed the specificity of the antibody. Double labeling with P2X7 and cell markers demonstrated that P2X7 was co-localized with Iba1, but not with NeuN or GFAP, although some P2X7-positive signals were observed near astrocytic processes.

To investigate the functional role of P2X7 in neuropathic pain, we administered a selective P2X7 antagonist intrathecally and found that it significantly reduced mechanical hypersensitivity in SNI rats compared to vehicle-treated controls. No effect was observed on the contralateral side.

Discussion

Recent evidence suggests that purinergic signaling in microglia is essential for their interaction with neurons. Microglia express various P2 receptors that respond to different concentrations of ATP. The upregulation of P2X4 and P2Y12 receptors in spinal microglia has been well established in nerve injury models. Our results support the involvement of P2X7 as well, through both gene and protein expression in microglia after nerve injury.

Systemic administration of the P2X7 antagonist A438079 was previously shown to have antinociceptive effects. Our findings confirm that local inhibition of P2X7 in the spinal cord also attenuates neuropathic pain. Although some expression of P2X7 was observed in astrocytes, the predominant expression was in microglia, suggesting a central role for microglial P2X7 in neuropathic pain.

P2X7 activation by ATP in vitro leads to the release of pro-inflammatory cytokines such as TNF-α and IL-1β. These cytokines may contribute to a pro-inflammatory environment in the spinal cord. P2X7 and P2X4 are also known to regulate transcriptional activity related to neurotrophic and inflammatory responses. Thus, activation of P2X7 by extracellular ATP may initiate signaling cascades that exacerbate neuropathic pain.

Conclusion

This study demonstrates that peripheral nerve injury induces the expression of P2X7 mRNA and protein in spinal microglia. The upregulation of P2X7 correlates with the development of mechanical hypersensitivity. Pharmacological inhibition of P2X7 reduces pain behavior, supporting its role in the pathogenesis of neuropathic pain. These findings suggest that spinal A-438079 microglial P2X7 receptors represent a promising target for therapeutic intervention in neuropathic pain.