Early Phase Of Neuropathic Pain Research Articles

Descending mechanism by which medial prefrontal cortex endocannabinoid signaling controls the development of neuropathic pain and neuronal activity of dorsal root ganglion.

Although regulation of nociceptive processes in the dorsal horn by deep brain structures has long been established, the role of cortical networks in pain regulation is minimally explored. The medial prefrontal cortex (mPFC) is a key brain area in pain processing that receives ascending nociceptive input and exerts top-down control of pain sensation. We have shown critical changes in mPFC synaptic function during neuropathic pain, controlled by endocannabinoid (eCB) signaling. This study tests whether mPFC eCB signaling modulates neuropathic pain through descending control. Intra-mPFC injection of cannabinoid receptor type 1 (CB1R) agonist WIN-55,212-2 (WIN) in the chronic phase transiently alleviates the pain-like behaviors in spared nerve injury (SNI) rats. By contrast, intra-mPFC injection of CB1R antagonist AM4113 in the early phase of neuropathic pain reduces the development of pain-like behaviors in the chronic phase. Spared nerve injury reduced the mechanical threshold to induce action potential firing of dorsal horn wide-dynamic-range neurons, but this was reversed in rats by WIN in the chronic phase of SNI and by mPFC injection of AM4113 in the early phase of SNI. Elevated dorsal root ganglion neuronal activity after injury was also diminished in rats by mPFC injection of AM4113, potentially by reducing antidromic activity and subsequent neuronal inflammation. These findings suggest that depending on the phase of the pain condition, both blocking and activating CB1 receptors in the mPFC can regulate descending control of pain and affect both dorsal horn neurons and peripheral sensory neurons, contributing to changes in pain sensitivity.

MMP-9 regulates CX3CL1/CX3CR1 in the early phase of neuropathic pain in chronic sciatic nerve constriction injury (CCI) rats.

To observe the effects of MMP-9 (matrix metalloproteases-9) on the mechanical allodynia and thermal hyperalgesia and the expression of CX3CL1 (CX3C chemokine ligand 1) protein in the spinal dorsal root ganglion (DRG) in rats with chronic sciatic nerve constriction injury (CCI). A total of 84 male SD rats were randomly divided into seven groups, namely the normal group , the sham operation group , the model group , the CCI + MMP-9 group, the CCI + TIMP-1 group , the CCI + siRNA group, and the CCI + MM-siRNA group. The CCI model was prepared 5 days after implantation of intrathecal catheter. The rat paw mechanical withdrawal threshold (PWMT) and paw thermal withdrawal latency (PWTL) were measured 1 day before CCI surgery and 1, 2, 3 and 5 days after CCI respectively. Western blot (WB) was used to detect the expressions of the MMP-9 and the CX3CL1 protein in the L5 DRG of the spinal cord 1 day after CCI operation. (I) Behavioral assessment of hyperalgesia: compared with the Sham group, the PWMT and PWTL of the CCI group were significantly reduced at each time point after CCI surgery; compared with the CCI group, the PWMT and PWTL of the CCI + MMP-9 group decreased 1 day after CCI; for the PWMT and PWTL of the CCI + TIMP-1 group and CCI + siRNA group, PWMT and PWTL increased 1 day after CCI; (II) The expressions of MMP-9 and CX3CL1 protein in the DRG of the spinal cord: compared with Sham group, the expressions of MMP-9 and CX3CL1 protein in the DRG of the CCI group increased significantly 1 day after CCI surgery; compared with the CCI group, the expressions increased in the CCI + MMP-9 group 1 day after CCI . However, the expressions of MMP-9 and CX3CL1 in the CCI + TIMP-1 group and CCI + siRNA group were reduced on the first postoperative day. The mechanism of MMP-9 participating in the early phase of neuropathic pain (NP) in CCI rats is related to the upregulation of CX3CL1.

Microglial TNFα Induces COX2 and PGI2 Synthase Expression in Spinal Endothelial Cells during Neuropathic Pain.

Prostaglandins (PGs) are typical lipid mediators that play a role in homeostasis and disease. They are synthesized from arachidonic acid by cyclooxygenase 1 (COX1) and COX2. Although COX2 has been reported to be upregulated in the spinal cord after nerve injury, its expression and functional roles in neuropathic pain remain unclear. In this study, we investigated the expression of Cox2, PGI2 synthase (Pgis), and prostaglandin I2 receptor (IP receptor) mRNA in the rat spinal cord after spared nerve injury (SNI). Levels of Cox2 and Pgis mRNA increased in endothelial cells from 24 to 48 h after nerve injury. IP receptor mRNA was constitutively expressed in dorsal horn neurons. A COX2 inhibitor and IP receptor antagonists attenuated pain behavior in the early phase of neuropathic pain. Furthermore, we examined the relationship between COX2 and tumor necrosis factor-α (TNFα) in the spinal cord of a rat SNI model. Levels of TNFα mRNA transiently increased in the spinal microglia 24 h after SNI. The TNF receptors Tnfr1 and Tnfr2 mRNA were colocalized with COX2. Intrathecal injection of TNFα induced Cox2 and Pgis mRNA expression in endothelial cells. These results revealed that microglia-derived TNFα induced COX2 and PGIS expression in spinal endothelial cells and that endothelial PGI2 played a critical role in neuropathic pain via neuronal IP receptor. These findings further suggest that the glia–endothelial cell interaction of the neurovascular unit via transient TNFα is involved in the generation of neuropathic pain.

Pain relief induces dopamine release in the rat nucleus accumbens during the early but not late phase of neuropathic pain

Comorbidity of chronic pain and depression has long been recognized in the clinic, and preclinical studies have reported depression-like behaviors in animal models of chronic pain. These findings suggest a common neuronal basis for chronic pain and depression. The neuronal pathway from the ventral tegmental area to the nucleus accumbens (NAc) is critical in the mesolimbic dopamine (DA) reward circuit, and dysfunction of this pathway has been implicated in depression. Although time-dependent development of depression-related behaviors has been reported in chronic pain animals, time-dependent functional changes in this pathway remain to be examined. To address this issue, we examined the effects of two types of rewards, pain relief by intrathecal injection of pregabalin (100μg in 10μL phosphate buffered saline) and 30% sucrose solution intake, on intra-NAc DA release in rats subjected to spinal nerve ligation (SNL). Specifically, the effects were investigated during the early (17–20days after ligation) and late (31–34days after ligation) phases of neuropathic pain. Pain relief increased the intra-NAc DA levels in the SNL rats during the early but not late phase of neuropathic pain. Intake of the sucrose solution increased the intra-NAc DA levels both in the SNL and sham animals during the early phase of neuropathic pain, while it induced DA release in the sham but not SNL animals during the late phase. These results suggest that dysfunction of the mesolimbic DA reward circuit develops in a time-dependent manner. Mesolimbic DA reward circuit dysfunction might be a common neuronal mechanism underlying chronic pain and depression, and a potential target for novel analgesic and antidepressant medications.

Macrophage-Colony Stimulating Factor Derived from Injured Primary Afferent Induces Proliferation of Spinal Microglia and Neuropathic Pain in Rats.

Peripheral nerve injury induces proliferation of microglia in the spinal cord, which can contribute to neuropathic pain conditions. However, candidate molecules for proliferation of spinal microglia after injury in rats remain unclear. We focused on the colony-stimulating factors (CSFs) and interleukin-34 (IL-34) that are involved in the proliferation of the mononuclear phagocyte lineage. We examined the expression of mRNAs for macrophage-CSF (M-CSF), granulocyte macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF) and IL-34 in the dorsal root ganglion (DRG) and spinal cord after spared nerve injury (SNI) in rats. RT-PCR and in situ hybridization revealed that M-CSF and IL-34, but not GM- or G-CSF, mRNAs were constitutively expressed in the DRG, and M-CSF robustly increased in injured-DRG neurons. M-CSF receptor mRNA was expressed in naive rats and increased in spinal microglia following SNI. Intrathecal injection of M-CSF receptor inhibitor partially but significantly reversed the proliferation of spinal microglia and in early phase of neuropathic pain induced by SNI. Furthermore, intrathecal injection of recombinant M-CSF induced microglial proliferation and mechanical allodynia. Here, we demonstrate that M-CSF is a candidate molecule derived from primary afferents that induces proliferation of microglia in the spinal cord and leads to induction of neuropathic pain after peripheral nerve injury in rats.

Mas-related gene (Mrg) C receptors inhibit mechanical allodynia and spinal microglia activation in the early phase of neuropathic pain in rats

Mas-related gene (Mrg) C receptors are exclusively expressed in the trigeminal and dorsal root ganglia (DRG). However, their functional roles are poorly understood. This study was aimed to determine the effect of MrgC receptors on pain hypersensitivity in the early phase of neuropathic pain and its underlying mechanisms. Intrathecal (i.t.) administration of the selective MrgC receptor agonist bovine adrenal medulla 8–22 (BAM8-22) at 1 or 10nmol attenuated mechanical allodynia one day after L5 spinal nerve ligation (SNL) surgery. I.t. BAM8-22 (10 nmol) inhibited SNL-induced microglia activation in the spinal dorsal horn on day 2 post-SNL. The BAM8-22 treatment also abolished SNL-induced upregulation of neuronal nitric oxide synthesis (nNOS) in the dorsal root ganglia (DRG). On the other hand, SNL, but not sham, surgery reduced the expression of MrgC receptor mRNA in the injured L5 DRG without changing thier levels in the adjacent uninjured L4 or L6 DRG on day 2 following the surgery. These results suggest that the activation of MrgC receptors can relieve pain hypersensitivity by the inhibition of nNOS increase in DRG neurons and microglia activation in the spinal dorsal horn in the early time following peripheral nerve injury. This study provides evidence that MrgC receptors could be targeted as a novel therapy for neuropathic pain with limited unwanted effects.

Activation of mGluR1 contributes to neuronal hyperexcitability in the rat anterior cingulate cortex via inhibition of HCN channels

Neuronal hyperexcitability in the anterior cingulate cortex (ACC) is considered as one of the most important pathological changes responsible for the chronification of neuropathic pain. However, the underlying mechanisms remain elusive. In the present study, we investigated the possible mechanisms using a rat model of chronic constriction injury (CCI) to the sciatic nerve. We found a substantial decrease in hyperpolarization-activated/cyclic nucleotide-gated (HCN) currents in layer 5 pyramidal neurons (L5 PNs) in ACC slices, which dramatically increased the excitability of these neurons. This effect could be mimicked in sham slices by activating group 1 metabotropic glutamate receptors, and be blocked in CCI slices by inhibiting metabotropic glutamate receptor subtype 1 (mGluR1). Next, the inhibition of HCN currents was reversed by a protein kinase C (PKC) inhibitor, followed by a reduced neuronal hyperexcitability. Furthermore, HCN channel subtype 1 (HCN1) level was significantly reduced after CCI, whereas mGluR1 level increased. These changes were mainly observed in L5 of the ACC, where HCN1 and mGluR1 were highly colocalized. For behavioral tests, intra-ACC microinjection of mGluR1-shRNA suppressed the CCI-induced behavioral hypersensitivity, particularly thermal hyperalgesia, but not aversive behavior, and this effect was attenuated by the pre-blockade of HCN channels. Taken together, the neuronal hyperexcitability of ACC L5 PNs likely results from an upregulation of mGluR1 and a downstream pathway involving PKC activation and a downregulation of HCN1 in the early phase of neuropathic pain. These alterations may at least in part contribute to the development of behavioral hypersensitivity in CCI rats.

The Macrophage-Mediated Effects of the Peroxisome Proliferator-Activated Receptor-Gamma Agonist Rosiglitazone Attenuate Tactile Allodynia in the Early Phase of Neuropathic Pain Development

Neuroinflammation triggered by macrophage infiltration into sites of peripheral nerve injury may result in neuropathic pain. Peroxisome proliferator-activated receptor (PPAR)γ signaling regulates the properties of macrophages. However, the macrophage-mediated effects of PPARγ signaling on neuropathic pain triggered by peripheral inflammation have not been investigated. To evaluate the peripheral effects of PPARγ signaling on tactile allodynia, we administered the blood-brain barrier-impermeant PPARγ agonist, rosiglitazone, after partial sciatic nerve ligation (PSNL) as (1) systemic treatment during different phases of neuropathic pain development, (2) local injection to the PSNL site in the early phase, or (3) peritoneal macrophages pretreated with rosiglitazone transplanted into the PSNL site. In addition, the direct effect of rosiglitazone was evaluated in peritoneal macrophages activated with interferon-γ. Systemic rosiglitazone treatment early in the course of progressive inflammation ameliorated tactile allodynia, macrophage infiltration, and production of proinflammatory mediators including cyclooxygenase-2, inducible nitric oxide synthase, and matrix metalloprotease 9 at the PSNL site. Local injection of rosiglitazone and transplantation of rosiglitazone-treated peritoneal macrophages at the ligation site significantly improved tactile allodynia. In peritoneal macrophages, rosiglitazone down-regulated interferon-γ-induced gene expression of cyclooxygenase-2 and inducible nitric oxide synthase and attenuated the chemotactic response to monocyte chemotactic protein-1. Rosiglitazone treatment in the early phase of neuropathic pain significantly alleviated the development of tactile allodynia by regulating macrophage infiltration and production of proinflammatory molecules at the inflamed site. Our results indicate that the activation of PPARγ signaling in macrophages during the early phase may suppress neuropathic pain development.

Rapid Synaptic Remodeling in the Adult Somatosensory Cortex following Peripheral Nerve Injury and Its Association with Neuropathic Pain

Structural and functional plastic changes in the primary somatosensory cortex (S1) have been observed following peripheral nerve injury that often leads to neuropathic pain, which is characterized by tactile allodynia. However, remodeling of cortical connections following injury has been believed to take months or years; this is not temporally correlated with the rapid development of allodynia and S1 hyperexcitability. Here we first report, by using long-term two-photon imaging of postsynaptic dendritic spines in living adult mice, that synaptic connections in the S1 are rewired within days following sciatic nerve ligation through phase-specific and size-dependent spine survival/growth. Spine turnover in the S1 area corresponding to the injured paw markedly increased during an early phase of neuropathic pain and was restored in a late phase of neuropathic pain, which was prevented by immediate local blockade of the injured nerve throughout the early phase. New spines that generated before nerve injury showed volume decrease after injury, whereas more new spines that formed in the early phase of neuropathic pain became persistent and substantially increased their volume during the late phase. Further, preexisting stable spines survived less following injury than controls, and such lost persistent spines were smaller in size than the surviving ones, which displayed long-term potentiation-like enlargement over weeks. These results suggest that peripheral nerve injury induces rapid and selective remodeling of cortical synapses, which is associated with neuropathic pain development, probably underlying, at least partially, long-lasting sensory changes in neuropathic subjects.

Increase in hemokinin-1 mRNA in the spinal cord during the early phase of a neuropathic pain state.

Substance P (SP), a representative member of the tachykinin family, is involved in nociception under physiological and pathological conditions. Recently, hemokinin-1 (HK-1) was identified as a new member of this family. Although HK-1 acts on NK(1) tachykinin receptors that are thought to be innate for SP, the roles of HK-1 in neuropathic pain are still unknown. Using rats that had been subjected to chronic constrictive injury (CCI) of the sciatic nerve as a neuropathic pain model, we examined the changes in expression of SP- and HK-1-encoding genes (TAC1 and TAC4, respectively) in the L4/L5 spinal cord and L4/L5 dorsal root ganglia (DRGs) in association with changes in pain-related behaviours in this neuropathic pain state. The TAC4 mRNA level was increased on the ipsilateral side of the dorsal spinal cord, but not in DRGs, at day 3 after CCI. In contrast, the TAC1 mRNA level was significantly increased in the DRGs at day 3 after CCI without any changes in the dorsal spinal cord. Analysis of a cultured microglial cell line revealed the presence of TAC4 mRNA in microglial cells. Minocycline, an inhibitor of microglial activation, blocked the increased expression of TAC4 mRNA after CCI and inhibited the associated pain-related behaviours and microglial activation in the spinal cord. The present results suggest that HK-1 expression is increased at least partly in activated microglial cells after nerve injury and is clearly involved in the early phase of neuropathic pain.

A comparison of spinal Iba1 and GFAP expression in rodent models of acute and chronic pain

The treatment of acute and chronic pain is still deficient. The modulation of glial cells may provide novel targets to treat pain. We hypothesize that astrocytes and microglia participate in the initiation and maintenance of both, acute surgical and chronic neuropathic pain. Rats underwent paw incision, L5 nerve exposure or L5 nerve transection surgery. Behavioral mechanical allodynia was assessed using von Frey filaments. Immunohistochemistry was performed using anti-ionized calcium binding adaptor protein, Iba-1 (microglia), and anti-Glial Fibrillary Acidic Protein, GFAP (astrocytes) on day 1, 4 and 7 after surgery. Following paw incision and at spinal L5 segment GFAP expression was increased in laminae I–II and Iba1 in deep laminae on day 1, in the entire dorsal horn on day 4 and dissipated on day 7 after paw incision in parallel with the allodynia. L5 nerve transection induced mechanical allodynia from day 1 to 7 which correlated with Iba-1 increases on day 1, 4 (entire dorsal horn) and day 7 after nerve injury (deep laminae of the dorsal horn) at spinal L5 segment. Conversely, GFAP increased at later time points from day 4 (deep laminae) and on day 7 (entire dorsal horn). Our data demonstrates that astrocytes (GFAP expression) play a role in the initiation of acute pain and the maintenance of chronic pain while Iba-1 increases closely correlated with the early phase of neuropathic pain. Iba1 and GFAP increased rostrally, at L3 segment, after paw incision (day 4) and only Iba1 increased following L5 nerve transection (day 7).

Distinct roles of matrix metalloproteases in the early- and late-phase development of neuropathic pain

Treatment of neuropathic pain, triggered by multiple insults to the nervous system, is a clinical challenge because the underlying mechanisms of neuropathic pain development remain poorly understood. Most treatments do not differentiate between different phases of neuropathic pain pathophysiology and simply focus on blocking neurotransmission, producing transient pain relief. Here, we report that early- and late-phase neuropathic pain development in rats and mice after nerve injury require different matrix metalloproteinases (MMPs). After spinal nerve ligation, MMP-9 shows a rapid and transient upregulation in injured dorsal root ganglion (DRG) primary sensory neurons consistent with an early phase of neuropathic pain, whereas MMP-2 shows a delayed response in DRG satellite cells and spinal astrocytes consistent with a late phase of neuropathic pain. Local inhibition of MMP-9 by an intrathecal route inhibits the early phase of neuropathic pain, whereas inhibition of MMP-2 suppresses the late phase of neuropathic pain. Further, intrathecal administration of MMP-9 or MMP-2 is sufficient to produce neuropathic pain symptoms. After nerve injury, MMP-9 induces neuropathic pain through interleukin-1beta cleavage and microglial activation at early times, whereas MMP-2 maintains neuropathic pain through interleukin-1beta cleavage and astrocyte activation at later times. Inhibition of MMP may provide a novel therapeutic approach for the treatment of neuropathic pain at different phases.