TRPM2 participates the transformation of acute pain to chronic pain during injury‐induced neuropathic pain

Transient receptor melastatin 2 (TRPM2) is a nonselective Ca²⁺‐permeable cation channel highly expressed in brain and other tissues. Studies showed that TRPM2 contributed to the induction of inflammatory cytokine and chemokine of immune cells, resulted in neuropathic pain. However, how TRPM2 regulates neuropathic pain is not clear. The sciatic nerve chronic constriction injury (CCI) rat model was used to induce chronic neuropathic pain. The RNA and protein level of TRPM2 was detected with real‐time PCR and western blot. SiRNA targeting TRPM2 was used to knockdown the expression of TRPM2. Reactive oxygen species (ROS) levels were determined using H2DCFDA assay and NO production was analyzed by measuring the accumulated level of its stable metabolite (nitrite). We found that CCI significantly increased TRPM2 expression in dorsal root ganglion and spinal cord. Knockdown TRPM2 in early phase after CCI alleviated injury‐induced neuropathic pain. Mechanistically, we demonstrated that TRPM2 knockdown drastically inhibited the iNOS expression and NO generation, with decreased ROS generation in CCI rat. TRPM2 participates in the transformation of acute pain to chronic pain during injury‐induced neuropathic pain, which might serve as a potential therapeutic target for neuropathic pain.     

Maladaptive dendritic spine remodeling contributes to diabetic neuropathic pain

Diabetic neuropathic pain imposes a huge burden on individuals and society, and represents a major public health problem. Despite aggressive efforts, diabetic neuropathic pain is generally refractory to available clinical treatments. A structure-function link between maladaptive dendritic spine plasticity and pain has been demonstrated previously in CNS and PNS injury models of neuropathic pain. Here, we reasoned that if dendritic spine remodeling contributes to diabetic neuropathic pain, then (1) the presence of malformed spines should coincide with the development of pain, and (2) disrupting maladaptive spine structure should reduce chronic pain. To determine whether dendritic spine remodeling contributes to neuropathic pain in streptozotocin (STZ)-induced diabetic rats, we analyzed dendritic spine morphology and electrophysiological and behavioral signs of neuropathic pain. Our results show changes in dendritic spine shape, distribution, and shape on wide-dynamic-range (WDR) neurons within lamina IV-V of the dorsal horn in diabetes. These diabetes-induced changes were accompanied by WDR neuron hyperexcitability and decreased pain thresholds at 4 weeks. Treatment with NSC23766 (N(6)-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride), a Rac1-specific inhibitor known to interfere with spine plasticity, decreased the presence of malformed spines in diabetes, attenuated neuronal hyperresponsiveness to peripheral stimuli, reduced spontaneous firing activity from WDR neurons, and improved nociceptive mechanical pain thresholds. At 1 week after STZ injection, animals with hyperglycemia with no evidence of pain had few or no changes in spine morphology. These results demonstrate that diabetes-induced maladaptive dendritic spine remodeling has a mechanistic role in neuropathic pain. Molecular pathways that control spine morphogenesis and plasticity may be promising future targets for treatment.     

Thrombospondin-4 contributes to spinal sensitization and neuropathic pain states

Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-gene chip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naive rats is sufficient to enhance the frequency of EPSCs in spinal dorsal horn neurons, suggesting an increased excitatory presynaptic input, and to cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal presynaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.     

Immune and inflammatory mechanisms in neuropathic pain

Tissue damage, inflammation or injury of the nervous system may result in chronic neuropathic pain characterised by increased sensitivity to painful stimuli (hyperalgesia), the perception of innocuous stimuli as painful (allodynia) and spontaneous pain. Neuropathic pain has been described in about 1% of the US population, is often severely debilitating and largely resistant to treatment. Animal models of peripheral neuropathic pain are now available in which the mechanisms underlying hyperalgesia and allodynia due to nerve injury or nerve inflammation can be analysed. Recently, it has become clear that inflammatory and immune mechanisms both in the periphery and the central nervous system play an important role in neuropathic pain. Infiltration of inflammatory cells, as well as activation of resident immune cells in response to nervous system damage, leads to subsequent production and secretion of various inflammatory mediators. These mediators promote neuroimmune activation and can sensitise primary afferent neurones and contribute to pain hypersensitivity. Inflammatory cells such as mast cells, neutrophils, macrophages and T lymphocytes have all been implicated, as have immune-like glial cells such as microglia and astrocytes. In addition, the immune response plays an important role in demyelinating neuropathies such as multiple sclerosis (MS), in which pain is a common symptom, and an animal model of MS-related pain has recently been demonstrated. Here, we will briefly review some of the milestones in research that have led to an increased awareness of the contribution of immune and inflammatory systems to neuropathic pain and then review in more detail the role of immune cells and inflammatory mediators.     

Visceral, inflammatory and neuropathic pain in glycine receptor alpha 3-deficient mice

The alpha3-subunit of strychnine-sensitive glycine receptors is an important modulator of the pain-sensitizing effects of spinal prostaglandin prostaglandin E(2). Mice deficient for alpha3-subunit of strychnine-sensitive glycine receptors lack the prostaglandin E(2)-induced inhibition of glycinergic neurotransmission and recover faster from inflammation-induced hyperalgesia. It, however, remains unclear whether alpha3-subunit of strychnine-sensitive glycine receptors plays a role in other pain models involving prostaglandin synthesis, such as chemically induced pain or neuropathic pain. In this paper, we show a reduction of acetic acid-induced writhing responses in the absence of alpha3-subunit of strychnine-sensitive glycine receptors, but no changes in formalin-induced pain. Furthermore, alpha3-subunit of strychnine-sensitive glycine receptors-deficient mice develop normal thermal hyperalgesia and tactile allodynia. Thus, alpha3-subunit of strychnine-sensitive glycine receptors is involved in the modulation of moderate inflammatory acetic acid-induced pain responses, but neither in formalin-induced pain nor in neuropathic pain.     

Integrin signaling in inflammatory and neuropathic pain in the rat

Many painful conditions are associated with alterations in the extracellular matrix (ECM) of affected tissues. While several integrins, the receptors for ECM proteins, are present on sensory neurons that mediate pain, the possible role of these cell adhesion molecules in inflammatory or neuropathic pain has not been explored. We found that the intradermal injection of peptide fragments of domains of laminin and fibronectin important for adhesive signaling selectively inhibited the hyperalgesia caused by prostaglandin E2 (PGE2) and epinephrine (EPI), respectively. The block of EPI hyperalgesia was mimicked by other peptides containing the RGD integrin-binding sequence. Monoclonal antibodies (mAbs) against the alpha1 or alpha3 integrin subunits, which participate in laminin binding, selectively blocked PGE2 hyperalgesia, while a mAb against the alpha5 subunit, which participates in fibronectin binding, blocked only EPI-induced hyperalgesia. A mAb against the beta1 integrin subunit, common to receptors for both laminin and fibronectin, inhibited hyperalgesia caused by both agents, as did the knockdown of beta1 integrin expression by intrathecal injection of antisense oligodeoxynucleotides. The laminin peptide, but not the fibronectin peptides, also reversibly abolished the longer lasting inflammatory hyperalgesia induced by carrageenan. Finally, the neuropathic hyperalgesia caused by systemic administration of the cancer chemotherapy agent taxol was reversibly inhibited by antisense knockdown of beta1 integrin. These results strongly implicate specific integrins in the maintenance of inflammatory and neuropathic hyperalgesia.     

The role of TLR2 in nerve injury-induced neuropathic pain is essentially mediated through macrophages in peripheral inflammatory response

Activation of macrophages/microglia via toll-like receptors (TLRs) plays an important role in inflammation and host defense against pathogens. Pathogen-associated molecular patterns bind TLRs, thereby triggering NF-κB signaling and production of proinflammatory cytokines. Recent data suggest that nonpathogenic molecules resulting from trauma can also trigger inflammation via TLRs. We sought to determine whether peripheral nerve injury could induce the expression of TLR2 on the site of injury-damaged nerves and/or in the central nervous system and to investigate whether TLR2 is necessary for the development of nerve injury-induced neuropathic pain. We observed a significant increase in TLR2, IκB-α, and TNF-α mRNAs in damaged nerves. Increased inflammation-related molecules were found essentially on ED1(+) macrophages. Expression of both IκB-α and TNF-α in peripheral injured nerves was reduced in TLR2 deficient mice where the recruitment of ED1(+) cells is significantly impaired. Although after peripheral nerve injury, spinal microglia became highly activated showing an increase in Iba-1 immunoreactivity and an enlargement of their cell bodies, neither TLR2 mRNA nor IκB-α mRNA was detected in activated microglia. Nerve injury-evoked spinal microglial activation was not significantly altered in TLR2 KO mice. Paw withdrawal threshold and latency in response to mechanical and heat stimuli, respectively, decreased shortly after nerve lesion in wild type mice. In TLR2 KO mice, nerve injury-induced thermal hyperalgesia was completely abolished contrary to that seen in wild-type mice, whereas mechanical allodynia was partially reduced. We suggest that TLR2 is necessary for the development of neuropathic pain and its contribution is more important in thermal hypersensitivity than that of mechanical allodynia.     

Tumor necrosis factor-alpha contributes to below-level neuropathic pain after spinal cord injury

OBJECTIVE: Our objective was to elucidate the mechanisms responsible for below-level pain after partial spinal cord injury (SCI). METHODS: We used lateral hemisection to model central neuropathic pain and herpes simplex viral (HSV) vector-mediated transfer of the cleaved soluble receptor for tumor necrosis factor-alpha (TNF-alpha) to evaluate the role of TNF-alpha in the pathogenesis of below-level pain. RESULTS: We found activation of microglia and increased expression of TNF-alpha below the level of the lesion in the lumbar spinal cord after T13 lateral hemisection that correlated with emergence of mechanical allodynia in the hind limbs of rats. Lumbar TNF-alpha had an apparent molecular weight of 27 kDa, consistent with the full-length transmembrane form of the protein (mTNF-alpha). Expression of the p55 TNF soluble receptor (sTNFRs) by HSV-mediated gene transfer resulted in reduced pain behavior and a decreased number of ED1-positive cells, as well as decreased phosphorylation of the p38 MAP kinase (p-p38) and diminished expression of mTNF-alpha in the dorsal horn. INTERPRETATION: These results suggest that expression of mTNF-alpha after injury is related to development of pain, and that reverse signaling through mTNF-alpha by sTNFR at that level reduces cellular markers of inflammatory response and pain-related behavior.     

Acute inflammatory and neuropathic pain in Lewis and Fischer rats

Inbred, histocompatible Lewis and Fischer 344 rats (LEW and FIS) have been used to identify an inverse relationship between hypothalamic-pituitary-adrenal (HPA) axis activity and susceptibility to autoimmune and chronic inflammatory disorders, with LEW showing blunted HPA axis activity and increased susceptibility toward the development of autoimmunity and chronic inflammation, and FIS showing the opposite relationship. In the present study, LEW and FIS were used to determine the relationship between HPA axis function and acute inflammatory pain (carrageenan-induced hindpaw inflammation) and neuropathic pain (partial sciatic nerve ligation; PSNL). The results showed that carrageenan-induced thermal and mechanical allodynia and hyperalgesia were greater in FIS than in LEW. Similarly, FIS showed more carrageenan-induced hindpaw swelling and higher levels of myeloperoxidase (a measure of neutrophils) in the carrageenan-inflamed hindpaw. After PSNL, LEW showed a profound mechanical allodynia and hyperalgesia, whereas mechanical sensitivity in FIS was unaltered. However, FIS, but not LEW, developed thermal allodynia and hyperalgesia after PSNL. These results provide strong evidence for a positive relationship between HPA axis activity and acute inflammatory pain. The results also support a relationship between HPA axis activity and neuropathic pain, but the relationship is complex and may depend on the pain assay.     

Interleukin-1alphabeta gene-deficient mice show reduced nociceptive sensitivity in models of inflammatory and neuropathic pain but not post-operative pain

The pro-inflammatory cytokine interleukin-1 (IL-1) has been implicated in both inflammatory processes and nociceptive neurotransmission. To further investigate the role of IL-1 in different pain states, gene-disrupted mice lacking both IL-1alpha and IL-1beta genes (IL-1alphabeta (-/-)) were characterized in inflammatory, neuropathic, and post-operative pain models. IL-1alphabeta (-/-) mice showed normal sensorimotor function as measured by the rotorod assay compared to control mice (BALB/c). Acute and persistent formalin-induced nocifensive behaviors were reduced by 20% in IL-1alphabeta (-/-) mice as compared to control mice. IL-1alphabeta (-/-) mice also showed reduced inflammatory thermal and mechanical hyperalgesia compared to controls following the intraplantar administration of carrageenan or complete Freund's adjuvant (CFA). The duration of inflammatory hyperalgesia was shortened in IL-1alphabeta (-/-) mice versus controls in the CFA model. In contrast, deletion of IL-1alphabeta did not change the extent or the duration of post-operative pain developing after skin incision of the hind paw. Finally, time to onset, duration, and magnitude of mechanical allodynia were reduced in two models of neuropathic pain, spinal nerve L5-L6 ligation and chronic constriction injury of the sciatic nerve, in IL-1alphabeta (-/-) mice versus controls. These results demonstrate that IL-1alphabeta modulates both the generation and the maintenance of inflammatory and chronic neuropathic pain and that IL-1 may modulate nociceptive sensitivity to a greater extent in conditions of chronic as compared to acute pain.     

Complement activation contributes to leukocyte recruitment and neuropathic pain following peripheral nerve injury in rats

Complement activation triggers inflammation and has been implicated in neurological diseases associated with pain. However, the role of complement in neuropathic pain has not been clearly defined. In this study, we tested whether complement is activated by partial ligation of the rat sciatic nerve, a widely used model of neuropathic pain, and whether complement activation or inhibition in peripheral nerve influences leukocyte recruitment and neuropathic pain. We found that C3 deposition significantly increased from 6 h to 7 days in the injured nerve and was associated with the extent of thermal hyperalgesia and mechanical allodynia. However, no deposition of the membrane attack complex was detected. Complement activation by endoneurial injection of aggregated rat immunoglobulin G into normal sciatic nerve produced significant thermal hyperalgesia and mechanical allodynia of the ipsilateral hindpaw at 2-7 days after injection. This was accompanied by increased deposition of C3 and recruitment of macrophages at 7 days following injection. Complement inhibition using systemic injections of soluble complement receptor 1 (AVANT Immunotherapeutics, Inc., Needham, USA) into rats markedly suppressed C3 deposition and T-cell and macrophage recruitment to the injured nerve, and produced significant alleviation of thermal hyperalgesia and mechanical allodynia. These results demonstrate that C3 activation in the nerve contributes to increased infiltration of inflammatory cells and to neuropathic pain behaviors following peripheral nerve injury. Complement inhibition may be a potential therapeutic treatment for neuropathic pain.     

Differential involvement of spinal protein kinase C and protein kinase A in neuropathic and inflammatory pain in mice

In the present study, we demonstrated the differential role of spinal protein kinases in neuropathic and inflammatory pain. Mice with sciatic nerve ligation exhibited a spinal protein kinase C (PKC)-dependent neuropathic pain-like state. In contrast, an intraplanter injection of inflammatory agent caused a protein kinase A (PKA)-related thermal hyperalgesia. These findings suggest that the substantial activation of spinal PKC and PKA may differentially contribute to the development of respective chronic pain-like state in mice.     

Ca2+/calmodulin-dependent protein kinase II in the spinal cord contributes to neuropathic pain in a rat model of mononeuropathy

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to subserve activity-dependent neuronal plasticity in the central nervous system. To examine in vivo the implication of spinal CaMKII activity in the generation and development of neuropathic pain after peripheral nerve injury, we used an animal model of mononeuropathy, the chronic constriction injury (CCI) model, in the rat. We found that, 3 days after CCI, the total CaMKII (tCaMKII) immunoreactivity increased in the superficial laminae of the spinal cord and this increase continued for up to 14 days. The immunoreactivity of phosphorylated CaMKII showed an increase from 1 day after CCI, which preceded the up-regulation of tCaMKII. A non-selective N-methyl-d-aspartate receptor antagonist, MK801, significantly attenuated the increase of tCaMKII and phosphorylated CaMKII. Moreover, intrathecal administration of an inhibitor of CaMKII, KN93, before the CCI surgery attenuated the development of thermal hyperalgesia and mechanical allodynia. In addition, KN93 significantly reduced the nociceptive behavior in phase II of the formalin test. These findings demonstrate that the activity of CaMKII in spinal neurons is elevated after peripheral nerve injury and may be involved in central sensitization. The alteration of CaMKII is considered to be a neuroplastic change that occurs in spinal neurons that contributes to neuropathic pain, suggesting the potential for the development of novel therapeutics for neuropathic pain that target CaMKII.     

A behavioral test paradigm to measure the aversive quality of inflammatory and neuropathic pain in rats

The present experiment assessed the aversive quality of neuropathic and inflammatory pain in rats. Compared to sham-treated animals, L5 ligated (neuropathic) and complete Freund's adjuvant (inflammatory)-treated animals displayed an initial period of escape followed by avoidance of a preferred location of the test chamber that was associated with mechanical stimulation of the hyperalgesic paw. The onset of the avoidance behavior occurred during the first 10-15 min of behavioral testing and was maximal at 30 min. It is concluded that animals find mechanical stimulation of the hyperalgesic paw aversive and that this behavioral test paradigm is an additional method that may be used to assess nociception in rat neuropathic and inflammatory models.     

Rac1-regulated dendritic spine remodeling contributes to neuropathic pain after peripheral nerve injury

Although prior studies have implicated maladaptive remodeling of dendritic spines on wide-dynamic range dorsal horn neurons as a contributor to pain after spinal cord injury, there have been no studies on dendritic spines after peripheral nerve injury. To determine whether dendritic spine remodeling contributes to neuronal hyperexcitability and neuropathic pain after peripheral nerve injury, we analyzed dendritic spine morphology and functional influence in lamina IV–V dorsal horn neurons after sham, chronic constriction injury (CCI) of the sciatic nerve, and CCI treatment with NSC23766, a selective inhibitor of Rac1, which has been implicated in dendritic spine development. 10 days after CCI, spine density increased with mature, mushroom-shaped spines preferentially distributed along dendritic branch regions closer to the cell body. Because spine morphology is strongly correlated with synaptic function and transmission, we recorded the response of single units to innocuous and noxious peripheral stimuli and performed behavioral assays for tactile allodynia and thermal hyperalgesia. Wide dynamic range dorsal horn neurons of CCI animals exhibited hyperexcitable responses to a range of stimuli. They also showed reduced nociceptive thresholds in the ipsilateral hind paw. 3-day treatment with NSC23766 significantly reduced post-CCI spine dimensions and densities, and attenuated injury-induced hyperexcitability. Drug treatment reduced behavioral measures of tactile allodynia, but not for thermal hyperalgesia. Together, our results demonstrate that peripheral nerve injury induces Rac1-regulated remodeling of dendritic spines on dorsal horn neurons, and suggest that this spine remodeling contributes to neuropathic pain.