Differential involvement of trigeminal transition zone and laminated subnucleus caudalis in orofacial deep and cutaneous hyperalgesia: the effects of interleukin-10 and glial inhibitors

Background

Understanding the underlying mechanisms of neuropathic pain caused by damage to the peripheral nervous system remains challenging and could lead to significantly improved therapies. Disturbance of homeostasis not only occurs at the site of injury but also extends to the spinal cord and brain involving various types of cells. Emerging data implicate neuroimmune interaction in the initiation and maintenance of chronic pain hypersensitivity.

Results

In this study, we sought to investigate the effects of TGF-β1, a potent anti-inflammatory cytokine, in alleviating nerve injury-induced neuropathic pain in rats. By using a well established neuropathic pain animal model (partial ligation of the sciatic nerve), we demonstrated that intrathecal infusion of recombinant TGF-β1 significantly attenuated nerve injury-induced neuropathic pain. TGF-β1 treatment not only prevents development of neuropathic pain following nerve injury, but also reverses previously established neuropathic pain conditions. The biological outcomes of TGF-β1 in this context are attributed to its pleiotropic effects. It inhibits peripheral nerve injury-induced spinal microgliosis, spinal microglial and astrocytic activation, and exhibits a powerful neuroprotective effect by preventing the induction of ATF3⁺ neurons following nerve ligation, consequently reducing the expression of chemokine MCP-1 in damaged neurons. TGF-β1 treatment also suppresses nerve injury-induced inflammatory response in the spinal cord, as revealed by a reduction in cytokine expression.

Conclusion

Our findings revealed that TGF-β1 is effective in the treatment of neuropathic by targeting both neurons and glial cells. We suggest that therapeutic agents such as TGF-β1 having multipotent effects on different types of cells could work in synergy to regain homeostasis in local spinal cord microenvironments, therefore contributing to attenuate neuropathic pain.     

A critical role of toll-like receptor 2 in nerve injury-induced spinal cord glial cell activation and pain hypersensitivity

The activation of spinal cord glial cells has been implicated in the development of neuropathic pain upon peripheral nerve injury. The molecular mechanisms underlying glial cell activation, however, have not been clearly elucidated. In this study, we found that damaged sensory neurons induce the expression of tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and inducible nitric-oxide synthase genes in spinal cord glial cells, which is implicated in the development of neuropathic pain. Studies using primary glial cells isolated from toll-like receptor 2 knock-out mice indicate that damaged sensory neurons activate glial cells via toll-like receptor 2. In addition, behavioral studies using toll-like receptor 2 knock-out mice demonstrate that the expression of toll-like receptor 2 is required for the induction of mechanical allodynia and thermal hyperalgesia due to spinal nerve axotomy. The nerve injury-induced spinal cord microglia and astrocyte activation is reduced in the toll-like receptor 2 knock-out mice. Similarly, the nerve injury-induced pro-inflammatory gene expression in the spinal cord is also reduced in the toll-like receptor 2 knock-out mice. These data demonstrate that toll-like receptor 2 contributes to the nerve injury-induced spinal cord glial cell activation and subsequent pain hypersensitivity.     

Antagonism of the Prokineticin System Prevents and Reverses Allodynia and Inflammation in a Mouse Model of Diabetes

Neuropathic pain is a severe diabetes complication and its treatment is not satisfactory. It is associated with neuroinflammation-related events that participate in pain generation and chronicization. Prokineticins are a new family of chemokines that has emerged as critical players in immune system, inflammation and pain. We investigated the role of prokineticins and their receptors as modulators of neuropathic pain and inflammatory responses in experimental diabetes. In streptozotocin-induced-diabetes in mice, the time course expression of prokineticin and its receptors was evaluated in spinal cord and sciatic nerves, and correlated with mechanical allodynia. Spinal cord and sciatic nerve pro- and anti-inflammatory cytokines were measured as protein and mRNA, and spinal cord GluR subunits expression studied. The effect of preventive and therapeutic treatment with the prokineticin receptor antagonist PC1 on behavioural and biochemical parameters was evaluated. Peripheral immune activation was assessed measuring macrophage and T-helper cytokine production. An up-regulation of the Prokineticin system was present in spinal cord and nerves of diabetic mice, and correlated with allodynia. Therapeutic PC1 reversed allodynia while preventive treatment blocked its development. PC1 normalized prokineticin levels and prevented the up-regulation of GluN2B subunits in the spinal cord. The antagonist restored the pro-/anti-inflammatory cytokine balance altered in spinal cord and nerves and also reduced peripheral immune system activation in diabetic mice, decreasing macrophage proinflammatory cytokines and the T-helper 1 phenotype. The prokineticin system contributes to altered sensitivity in diabetic neuropathy and its inhibition blocked both allodynia and inflammatory events underlying disease.     

Spatial and temporal relationship between monocyte chemoattractant protein-1 expression and spinal glial activation following peripheral nerve injury

Peripheral nerve injury can induce spinal microglial/astrocyte activation. Substances released by activated glial cells excite spinal nociceptive neurons. Pharmacological disruption of glial activation or antagonism of substances released by activated glia prevent or reverse pain hypersensitivity. It is not known, however, what causes spinal cord glia to shift from a resting to an activated state. In an attempt to understand the potential role of monocyte chemoattractant protein-1 (MCP-1) in triggering spinal glial activation and its contribution to the development of neuropathic pain, we investigated the effect of peripheral nerve injury on MCP-1 expression in dorsal root ganglia (DRG) and the spinal cord, and established its temporal relationship with activation of spinal microglia and astrocytes. We observed that MCP-1 was induced by chronic constriction of the sciatic nerve in DRG sensory neurons, spinal cord motor neurons and in the superficial dorsal horn, ipsilateral to the injury. Neuronal MCP-1 induction was followed by surrounding microglial activation. After peaking at day 7 after injury, MCP-1 levels began to decline rapidly and had returned to baseline by day 150. In contrast, microglial activation peaked by day 14 and declined afterwards to reach a lower, yet significantly raised level beyond day 22 and remained increased until the end of the test period. Astrocyte activation became detectable later, progressed more slowly and also remained increased until the end of the test period, in parallel with a decreased nociceptive threshold. Our results suggest that neuronal MCP-1 may serve as a trigger for spinal microglial activation, which participates in the initiation of neuropathic pain. Delayed, sustained astrocyte activation may participate with microglia in the persistent phase of pain hypersensitivity.     

Toll-like receptor 3 contributes to spinal glial activation and tactile allodynia after nerve injury

Toll-like receptors (TLRs) play an essential role in innate immune responses and in the initiation of adaptive immune responses. Microglia, the resident innate immune cells in the CNS, express TLRs. In this study, we show that TLR3 is crucial for spinal cord glial activation and tactile allodynia after peripheral nerve injury. Intrathecal administration of TLR3 antisense oligodeoxynucleotide suppressed nerve injury-induced tactile allodynia, and decreased the phosphorylation of p38 mitogen-activated protein kinase, but not extracellular signal-regulated protein kinases 1/2, in spinal glial cells. Antisense knockdown of TLR3 also attenuated the activation of spinal microglia, but not astrocytes, caused by nerve injury. Furthermore, down-regulation of TLR3 inhibited nerve injury-induced up-regulation of spinal pro-inflammatory cytokines, such as interleukin-1β, interleukin-6, and tumor necrosis factor-α. Conversely, intrathecal injection of the TLR3 agonist polyinosine–polycytidylic acid induced behavioral, morphological, and biochemical changes similar to those observed after nerve injury. Indeed, TLR3-deficient mice did not develop tactile allodynia after nerve injury or polyinosine–polycytidylic acid injection. Our results indicate that TLR3 has a substantial role in the activation of spinal glial cells and the development of tactile allodynia after nerve injury. Thus, blocking TLR3 in the spinal glial cells might provide a fruitful strategy for treating neuropathic pain.     

MiR‐150 alleviates neuropathic pain via inhibiting toll‐like receptor 5

MicroRNAs (miRNAs) are reported as vital participators in the pathophysiological course of neuropathic pain. However, the underlying mechanisms of the functional roles of miRNAs in neuropathic pain are largely unknown. This study was designed to explore the potential role of miR‐150 in regulating the process of neuropathic pain in a rat model established by chronic sciatic nerve injury (CCI). Overexpression of miR‐150 greatly alleviated neuropathic pain development and reduced inflammatory cytokine expression, including COX‐2, interleukin IL‐6, and tumor necrosis factor (TNF)‐α in CCI rats. By bioinformatic analysis, 3′‐untranslated region (UTR) of Toll‐like receptor (TLR5) was predicted to be a target of miR‐150. TLR5 commonly serves as an important regulator of inflammation. Overexpression of miR‐150 significantly suppressed the expression of TLR5 in vitro and in vivo. Furthermore, upregulation of TLR5 decreased the miR‐150 expression and downregulation of TLR5 increased miR‐150, respectively. Overexpression of TLR5 significantly reversed the miR‐150‐induced suppressive effects on neuropathic pain. In conclusion, our current study indicates that miR‐150 may inhibit neuropathic pain development of CCI rats through inhibiting TLR5‐mediated neuroinflammation. Our findings suggest that miR‐150 may provide a novel therapeutic target for neuropathic pain treatment.     

The critical role of spinal ceramide in the development of partial sciatic nerve ligation-induced neuropathic pain in mice

Recent observations indicate that peripheral nerve injury induces central sensitization through microglial activation and the release of inflammatory cytokines, resulting in the development of neuropathic pain. However, the underlying mechanisms of this phenomenon remain to be fully elucidated. In this study, we examined the involvement of spinal ceramide, a bioactive lipid, in the development of neuropathic pain induced by partial sciatic nerve ligation (PSL). We found that the mRNA expression levels for ceramide synthase and neutral sphingomyelinase, which are enzymes of ceramide biosynthesis, were up-regulated in the spinal cord from 3h to 1 day after PSL. The mRNA expressions of cytokines (interleukin-1β and tumor necrosis factor-α) and the microglial specific molecules (Iba-1 and CD11b) were also increased in the spinal cord after PSL. In the von Frey test, intrathecal injection of the ceramide biosynthesis inhibitors Fumonisin B1 and GW4869 at 3h and day 3 after PSL significantly attenuated PSL-induced tactile allodynia. By immunohistochemistry, microglial activation in the dorsal horn was suppressed by Fumonisin B1 and GW4869. Therefore, we conclude that spinal ceramide may play a crucial role in PSL-induced neuropathic pain through the activation of microglia.     

Involvement of TRPM2 in Peripheral Nerve Injury-Induced Infiltration of Peripheral Immune Cells into the Spinal Cord in Mouse Neuropathic Pain Model

Recent evidence suggests that transient receptor potential melastatin 2 (TRPM2) expressed in immune cells plays an important role in immune and inflammatory responses. We recently reported that TRPM2 expressed in macrophages and spinal microglia contributes to the pathogenesis of inflammatory and neuropathic pain aggravating peripheral and central pronociceptive inflammatory responses in mice. To further elucidate the contribution of TRPM2 expressed by peripheral immune cells to neuropathic pain, we examined the development of peripheral nerve injury-induced neuropathic pain and the infiltration of immune cells (particularly macrophages) into the injured nerve and spinal cord by using bone marrow (BM) chimeric mice by crossing wildtype (WT) and TRPM2-knockout (TRPM2-KO) mice. Four types of BM chimeric mice were prepared, in which irradiated WT or TRPM2-KO recipient mice were transplanted with either WT-or TRPM2-KO donor mouse-derived green fluorescence protein-positive (GFP⁺) BM cells (TRPM2^BM+/Rec+, TRPM2^BM–/Rec+, TRPM2^BM+/Rec–, and TRPM2^BM–/Rec– mice). Mechanical allodynia induced by partial sciatic nerve ligation observed in TRPM2^BM+/Rec+ mice was attenuated in TRPM2^BM–/Rec+, TRPM2^BM+/Rec–, and TRPM2^BM–/Rec– mice. The numbers of GFP⁺ BM-derived cells and Iba1/GFP double-positive macrophages in the injured sciatic nerve did not differ among chimeric mice 14 days after the nerve injury. In the spinal cord, the number of GFP⁺ BM-derived cells, particularly GFP/Iba1 double-positive macrophages, was significantly decreased in the three TRPM2-KO chimeric mouse groups compared with TRPM2^BM+/Rec+ mice. However, the numbers of GFP^–/Iba1⁺ resident microglia did not differ among chimeric mice. These results suggest that TRPM2 plays an important role in the infiltration of peripheral immune cells, particularly macrophages, into the spinal cord, rather than the infiltration of peripheral immune cells into the injured nerves and activation of spinal-resident microglia. The spinal infiltration of macrophages mediated by TRPM2 may contribute to the pathogenesis of neuropathic pain.     

Differential Changes in Neuronal Excitability in the Spinal Dorsal Horn After Spinal Nerve Ligation in Rats

Previous studies demonstrated that peripheral nerve injury induced excessive neuronal response and glial activation in the spinal cord dorsal horn, and such change has been proposed to reflect the development and maintenance of neuropathic pain states. The aim of this study was to examine neuronal excitability and glial activation in the spinal dorsal horn after peripheral nerve injury. We examined noxious heat stimulation-induced c-Fos protein-like immunoreactivity (Fos-LI) neuron profiles in fourth-to-sixth lumbar (L4–L6) level spinal dorsal horn neurons after fifth lumbar spinal nerve ligation (L5 SNL). Immunofluorescence labeling of OX-42 and GFAP was also performed in histological sections of the spinal cord. A significant increase in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L4 level was found at 3 days after SNL, but returned to a level similar to that in sham-operated controls by 14 days after injury. As expected, a decrease in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L5 level was found at 3 days after SNL. However, these profiles had reappeared in large numbers by 14 and 21 days after injury. Immunofluorescence labeling of OX-42 and GFAP indicated sequential activation of microglia and astrocytes in the spinal dorsal horn. We conclude that nerve injury causes differential changes in neuronal excitability in the spinal dorsal horn, which may coincide with glial activation. These changes may play a substantial role in the pathogenesis of neuropathic pain after peripheral nerve injury.     

Losartan attenuates neuroinflammation and neuropathic pain in paclitaxel‐induced peripheral neuropathy

Paclitaxel‐induced peripheral neuropathy (PIPN) is often associated with neuropathic pain and neuroinflammation in the central and peripheral nervous system. Antihypertensive drug losartan, an angiotensin II receptor type 1 (AT1R) blocker, was shown to have anti‐inflammatory and neuroprotective effects in disease models, predominantly via activation of peroxisome proliferator‐activated receptor gamma (PPARγ). Here, the effect of systemic losartan treatment (100 mg/kg/d) on mechanical allodynia and neuroinflammation was evaluated in rat PIPN model. The expression of pro‐inflammatory markers protein and mRNA levels in dorsal root ganglia (DRGs) and spinal cord dorsal horn (SCDH) were measured with Western blot, ELISA and qPCR 10 and 21 days after PIPN induction. Losartan treatment attenuated mechanical allodynia significantly. Paclitaxel induced overexpression of C‐C motif chemokine ligand 2 (CCL2), tumour necrosis alpha (TNFα) and interleukin‐6 (IL‐6) in DRGs, where the presence of macrophages was demonstrated. Neuroinflammatory changes in DRGs were accompanied with glial activation and pro‐nociceptive modulators production in SCDH. Losartan significantly attenuated paclitaxel‐induced neuroinflammatory changes and induced expression of pro‐resolving markers (Arginase 1 and IL‐10) indicating a possible shift in macrophage polarization. Considering the safety profile of losartan, acting also as partial PPARγ agonist, it may be considered as a novel treatment strategy for PIPN patients.     

Proteome of synaptosome‐associated proteins in spinal cord dorsal horn after peripheral nerve injury

Peripheral nerve injury may lead to neuroadaptive changes of cellular signals in spinal cord that are thought to contribute to central mechanisms underlying neuropathic pain. Here we used a 2‐DE‐based proteomic technique to determine the global expression changes of synaptosome‐associated proteins in spinal cord dorsal horn after unilateral fifth spinal nerve injury (SNI). The fifth lumbar dorsal horns ipsilateral to SNI or sham surgery were harvested on day 14 post‐surgery, and the total soluble and synaptosomal fractions were isolated. The proteins derived from the synaptosomal fraction were resolved by 2‐DE. We identified 27 proteins that displayed different expression levels after SNI, including proteins involved in transmission and modulation of noxious information, cellular metabolism, membrane receptor trafficking, oxidative stress, apoptosis, and degeneration. Six of the 27 proteins were chosen randomly and further validated in the synaptosomal fraction by Western blot analysis. Unexpectedly, Western blot analysis showed that only one protein in the total soluble fraction exhibited a significant expression change after SNI. The data indicate that peripheral nerve injury changes not only protein expression but also protein subcellular distribution in dorsal horn cells. These changes might participate in the central mechanism that underlies the maintenance of neuropathic pain.     

Genetic evidence for an essential role of neuronally expressed IL-6 signal transducer gp130 in the induction and maintenance of experimentally induced mechanical hypersensitivity in vivo and in vitro

Background

A preconditioning stimulus can trigger a neuroprotective phenotype in the nervous system - a preconditioning nerve lesion causes a significant increase in axonal regeneration, and cerebral preconditioning protects against subsequent ischemia. We hypothesized that a preconditioning nerve lesion induces gene/protein modifications, neuronal changes, and immune activation that may affect pain sensation following subsequent nerve injury. We examined whether a preconditioning lesion affects neuropathic pain and neuroinflammation after peripheral nerve injury.

Results

We found that a preconditioning crush injury to a terminal branch of the sciatic nerve seven days before partial ligation of the sciatic nerve (PSNL; a model of neuropathic pain) induced a significant attenuation of pain hypersensitivity, particularly mechanical allodynia. A preconditioning lesion of the tibial nerve induced a long-term significant increase in paw-withdrawal threshold to mechanical stimuli and paw-withdrawal latency to thermal stimuli, after PSNL. A preconditioning lesion of the common peroneal induced a smaller but significant short-term increase in paw-withdrawal threshold to mechanical stimuli, after PSNL. There was no difference between preconditioned and unconditioned animals in neuronal damage and macrophage and T-cell infiltration into the dorsal root ganglia (DRGs) or in astrocyte and microglia activation in the spinal dorsal and ventral horns.

Conclusions

These results suggest that prior exposure to a mild nerve lesion protects against adverse effects of subsequent neuropathic injury, and that this conditioning-induced inhibition of pain hypersensitivity is not dependent on neuroinflammation in DRGs and spinal cord. Identifying the underlying mechanisms may have important implications for the understanding of neuropathic pain due to nerve injury.     

The spinal cord expression of neuronal and inducible nitric oxide synthases and their contribution in the maintenance of neuropathic pain in mice

Background

Nitric oxide generated by neuronal (NOS1), inducible (NOS2) or endothelial (NOS3) nitric oxide synthases contributes to pain processing, but the exact role of NOS1 and NOS2 in the maintenance of chronic peripheral neuropathic pain as well as the possible compensatory changes in their expression in the spinal cord of wild type (WT) and NOS knockout (KO) mice at 21 days after total sciatic nerve ligation remains unknown.

Methodology/Principal Findings

The mechanical and thermal allodynia as well as thermal hyperalgesia induced by sciatic nerve injury was evaluated in WT, NOS1-KO and NOS2-KO mice from 1 to 21 days after surgery. The mRNA and protein levels of NOS1, NOS2 and NOS3 in the spinal cord of WT and KO mice, at 21 days after surgery, were also assessed. Sciatic nerve injury led to a neuropathic syndrome in WT mice, in contrast to the abolished mechanical allodynia and thermal hyperalgesia as well as the decreased or suppressed thermal allodynia observed in NOS1-KO and NOS2-KO animals, respectively. Sciatic nerve injury also increases the spinal cord expression of NOS1 and NOS2 isoforms, but not of NOS3, in WT and NOS1-KO mice respectively. Moreover, the presence of NOS2 is required to increase the spinal cord expression of NOS1 whereas an increased NOS1 expression might avoid the up-regulation of NOS2 in the spinal cord of nerve injured WT mice.

Conclusions/Significance

These data suggest that the increased spinal cord expression of NOS1, regulated by NOS2, might be responsible for the maintenance of chronic peripheral neuropathic pain in mice and propose these enzymes as interesting therapeutic targets for their treatment.     

The glial modulatory drug AV411 attenuates mechanical allodynia in rat models of neuropathic pain

Controlling neuropathic pain is an unmet medical need and we set out to identify new therapeutic candidates. AV411 (ibudilast) is a relatively nonselective phosphodiesterase inhibitor that also suppresses glial-cell activation and can partition into the CNS. Recent data strongly implicate activated glial cells in the spinal cord in the development and maintenance of neuropathic pain. We hypothesized that AV411 might be effective in the treatment of neuropathic pain and, hence, tested whether it attenuates the mechanical allodynia induced in rats by chronic constriction injury (CCI) of the sciatic nerve, spinal nerve ligation (SNL) and the chemotherapeutic paclitaxel (Taxol). Twice-daily systemic administration of AV411 for multiple days resulted in a sustained attenuation of CCI-induced allodynia. Reversal of allodynia was of similar magnitude to that observed with gabapentin and enhanced efficacy was observed in combination. We further show that multi-day AV411 reduces SNL-induced allodynia, and reverses and prevents paclitaxel-induced allodynia. Also, AV411 cotreatment attenuates tolerance to morphine in nerve-injured rats. Safety pharmacology, pharmacokinetic and initial mechanistic analyses were also performed. Overall, the results indicate that AV411 is effective in diverse models of neuropathic pain and support further exploration of its potential as a therapeutic agent for the treatment of neuropathic pain.     

Role of Lipocalin-2-Chemokine Axis in the Development of Neuropathic Pain following Peripheral Nerve Injury

Lipocalin 2 (LCN2), which is also known as 24p3 and neutrophil gelatinase-associated lipocalin (NGAL), binds small, hydrophobic ligands and interacts with cell surface receptor 24p3R to regulate diverse cellular processes. In the present study, we examined the role of LCN2 in the pathogenesis of neuropathic pain using a mouse model of spared nerve injury (SNI). Lcn2 mRNA levels were significantly increased in the dorsal horn of the spinal cord after SNI, and LCN2 protein was mainly localized in neurons of the dorsal and ventral horns. LCN2 receptor 24p3R was expressed in spinal neurons and microglia after SNI. Lcn2-deficient mice exhibited significantly less mechanical pain hypersensitivity during the early phase after SNI, and an intrathecal injection of recombinant LCN2 protein elicited mechanical pain hypersensitivity in naive animals. Lcn2 deficiency, however, did not affect acute nociceptive pain. Lcn2-deficient mice showed significantly less microglial activation and proalgesic chemokine (CCL2 and CXCL1) production in the spinal cord after SNI than wild-type mice, and recombinant LCN2 protein induced the expression of these chemokines in cultured neurons. Furthermore, the expression of LCN2 and its receptor was detected in neutrophils and macrophages in the sciatic nerve following SNI, suggesting the potential role of peripheral LCN2 in neuropathic pain. Taken together, our results indicate that LCN2 plays a critical role in the development of pain hypersensitivity following peripheral nerve injury and suggest that LCN2 mediates neuropathic pain by inducing chemokine expression and subsequent microglial activation.     

Persistent At-Level Thermal Hyperalgesia and Tactile Allodynia Accompany Chronic Neuronal and Astrocyte Activation in Superficial Dorsal Horn following Mouse Cervical Contusion Spinal Cord Injury

In humans, sensory abnormalities, including neuropathic pain, often result from traumatic spinal cord injury (SCI). SCI can induce cellular changes in the CNS, termed central sensitization, that alter excitability of spinal cord neurons, including those in the dorsal horn involved in pain transmission. Persistently elevated levels of neuronal activity, glial activation, and glutamatergic transmission are thought to contribute to the hyperexcitability of these dorsal horn neurons, which can lead to maladaptive circuitry, aberrant pain processing and, ultimately, chronic neuropathic pain. Here we present a mouse model of SCI-induced neuropathic pain that exhibits a persistent pain phenotype accompanied by chronic neuronal hyperexcitability and glial activation in the spinal cord dorsal horn. We generated a unilateral cervical contusion injury at the C5 or C6 level of the adult mouse spinal cord. Following injury, an increase in the number of neurons expressing ΔFosB (a marker of chronic neuronal activation), persistent astrocyte activation and proliferation (as measured by GFAP and Ki67 expression), and a decrease in the expression of the astrocyte glutamate transporter GLT1 are observed in the ipsilateral superficial dorsal horn of cervical spinal cord. These changes have previously been associated with neuronal hyperexcitability and may contribute to altered pain transmission and chronic neuropathic pain. In our model, they are accompanied by robust at-level hyperaglesia in the ipsilateral forepaw and allodynia in both forepaws that are evident within two weeks following injury and persist for at least six weeks. Furthermore, the pain phenotype occurs in the absence of alterations in forelimb grip strength, suggesting that it represents sensory and not motor abnormalities. Given the importance of transgenic mouse technology, this clinically-relevant model provides a resource that can be used to study the molecular mechanisms contributing to neuropathic pain following SCI and to identify potential therapeutic targets for the treatment of chronic pathological pain.     

Selective activation of microglia in spinal cord but not higher cortical regions following nerve injury in adult mouse

Neuronal plasticity along the pathway for sensory transmission including the spinal cord and cortex plays an important role in chronic pain, including inflammatory and neuropathic pain. While recent studies indicate that microglia in the spinal cord are involved in neuropathic pain, a systematic study has not been performed in other regions of the central nervous system (CNS). In the present study, we used heterozygous Cx3cr1 ^GFP/+mice to characterize the morphological phenotypes of microglia following common peroneal nerve (CPN) ligation. We found that microglia showed a uniform distribution throughout the CNS, and peripheral nerve injury selectively activated microglia in the spinal cord dorsal horn and related ventral horn. In contrast, microglia was not activated in supraspinal regions of the CNS, including the anterior cingulate cortex (ACC), prefrontal cortex (PFC), primary and secondary somatosensory cortex (S1 and S2), insular cortex (IC), amygdala, hippocampus, periaqueductal gray (PAG) and rostral ventromedial medulla (RVM). Our results provide strong evidence that nerve injury primarily activates microglia in the spinal cord of adult mice, and pain-related cortical plasticity is likely mediated by neurons.     

Peripheral Nerve Lesion Induces an Up-regulation of Spy1 in Rat Spinal Cord

Spy1, as a member of the Speedy/RINGO family and a novel activator of cyclin-dependent kinases, was shown to promote cell cycle progression and cell survival in response to DNA damage. While its expression and roles in nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve injury model in adult rats and studied the dynamic changes of Spy1 expression in lumbar spinal cord. Temporally, Spy1 expression was increased shortly after sciatic nerve crush and peaked at day 2. Spatially, Spy1 was widely expressed in the lumbar spinal cord including neurons and glial cells. While after injury, Spy1 expression was increased predominantly in astrocytes and microglia, which were largely proliferated. Moreover, there was a concomitant up-regulation of CDK2 activity and down-regulation of p27. Collectively, we hypothesized peripheral nerve injury induced an up-regulation of Spy1 in lumbar spinal cord, which was associated with glial proliferation. Ye Huang and Yonghua Liu contributed equally to this work.     

Roles of extracellular signal-regulated protein kinases 5 in spinal microglia and primary sensory neurons for neuropathic pain

Neuropathic pain that occurs after peripheral nerve injury is poorly controlled by current therapies. Increasing evidence shows that mitogen-activated protein kinase (MAPK) play an important role in the induction and maintenance of neuropathic pain. Here we show that activation of extracellular signal-regulated protein kinases 5 (ERK5), also known as big MAPK1, participates in pain hypersensitivity caused by nerve injury. Nerve injury increased ERK5 phosphorylation in spinal microglia and in both damaged and undamaged dorsal root ganglion (DRG) neurons. Antisense knockdown of ERK5 suppressed nerve injury-induced neuropathic pain and decreased microglial activation. Furthermore, inhibition of ERK5 blocked the induction of transient receptor potential channels and brain-derived neurotrophic factor expression in DRG neurons. Our results show that ERK5 activated in spinal microglia and DRG neurons contributes to the development of neuropathic pain. Thus, blocking ERK5 signaling in the spinal cord and primary afferents has potential for preventing pain after nerve damage.     

Effective neuropathic pain relief through sciatic nerve administration of GAD65-expressing rAAV2

Recently, we demonstrated that the administration of GAD65-expressing rAAV2 to DRG attenuates peripheral neuropathy by inducing GABA release in the spinal cord. However, the direct injection to DRG is invasive and may therefore cause nerve injury and other side effects.To circumvent this surgical intervention, we explored the potential of a much simpler and less invasive route of sciatic nerve administration. Using a neuropathic pain model, we introduced rAAV2-GAD65 through sciatic nerve and examined its therapeutic potency in pain-related behavior tests. Both GFP and GAD65 expression indicated that effective transgene delivery to the DRG can be accomplished via sciatic nerve administration. Equally importantly, the GABA concentration in the spinal cord increased significantly after GAD65 introduction, and pain symptoms were dramatically reduced and persistently controlled. The implication is that the sciatic nerve is a highly promising route for delivering rAAV2 to the DRG, and thus represents a much less invasive, clinically viable gene therapy option.