Identification of tetracycline combinations as EphB1 tyrosine kinase inhibitors for treatment of neuropathic pain.

Previous studies have demonstrated that the synaptic EphB1 receptor tyrosine kinase is a major mediator of neuropathic pain, suggesting that targeting the activity of this receptor might be a viable therapeutic option. Therefore, we set out to determine if any FDA-approved drugs can act as inhibitors of the EphB1 intracellular catalytic domain. An in silico screen was first used to identify a number of tetracycline antibiotics which demonstrated potential docking to the ATP-binding catalytic domain of EphB1. Kinase assays showed that demeclocycline, chlortetracycline, and minocycline inhibit EphB1 kinase activity at low micromolar concentrations. In addition, we cocrystallized chlortetracycline and EphB1 receptor, which confirmed its binding to the ATP-binding domain. Finally, in vivo administration of the three-tetracycline combination inhibited the phosphorylation of EphB1 in the brain, spinal cord, and dorsal root ganglion (DRG) and effectively blocked neuropathic pain in mice. These results indicate that demeclocycline, chlortetracycline, and minocycline can be repurposed for treatment of neuropathic pain and potentially for other indications that would benefit from inhibition of EphB1 receptor kinase activity.

Human safety study of a selective neuronal adenylate cyclase 1 inhibitor NB001 which relieves the neuropathic pain and blocks ACC in adult mice.

Calcium-dependent, neuronal adenylyl cyclase subtype 1 (AC1) is critical for cortical potentiation and chronic pain. NB001 is a first-in-class drug acting as a selective inhibitor against AC1. The present study delineated the pharmacokinetic (PK) properties of human-used NB001 (hNB001) formulated as immediate-release tablet. This first-in-human (FIH) study was designed as randomized, double-blind, placebo-controlled trial. hNB001 showed placebo-like safety and good tolerability in healthy volunteers. A linear dose-exposure relationship was demonstrated at doses between 20 mg and 400 mg. The relatively small systemic exposure of hNB001 in human showed low bioavailability of this compound through oral administration, which can be improved through future dosage research. Food intake had minimal impact on the absorption of hNB001 tablet. Animal experiments further confirmed that hNB001 had strong analgesic effect in animal models of neuropathic pain. In brain slice prepared from the anterior cingulate cortex (ACC), bath application of hNB001 blocked the induction of long-term potentiation (LTP). These results from both rodents and human strongly suggest that hNB001 can be safely used for the future treatment of different types of chronic pain in human patients.

PCC-0105002, a novel small molecule inhibitor of PSD95-nNOS protein-protein interactions, attenuates neuropathic pain and corrects motor disorder associated with neuropathic pain model.

In view of postsynaptic density 95kDA (PSD95) tethers neuronal NO synthase (nNOS) to N-methyl-d-aspartate receptor (NMDAR), the PSD95-nNOS complex represents a therapeutic target of neuropathic pain. This study therefore sought to explore the ability of PCC-0105002, a novel PSD95-nNOS small molecule inhibitor, to alter pain sensitivity in rodent neuropathic pain models. Firstly, the IC50 of PCC-0105002 for PSD95 and NOS1 binding activity was determined using an Alpha Screen assay kit. Then, we examined the effects of PCC-0105002 in the mouse formalin test and in the rat spinal nerve ligation (SNL) model, and explored the ability of PCC-0105002 to mediate analgesia and to effect motor coordination in a rota-rod test. Moreover, the mechanisms whereby PCC-0105002 mediates analgesia was explored via western blotting, Golgi staining, and co-immunoprecipitation experiments in dorsal horn. The outcomes indicated that PCC-0105002 exhibited dose-dependent attenuation of phase II pain-associated behaviors in the formalin test. The result indicated that PCC-0105002 disrupted the PSD95-nNOS interaction with IC50 of 1.408 µM. In the SNL model, PCC-0105002 suppressed mechanical allodynia, thermal hyperalgesia, and abnormal dorsal horn wide dynamic range neuron discharge. PCC-0105002 mediated an analgesic effect comparable to that of MK-801, while it was better able to enhance motor coordination as compared with MK-801. Moreover, PCC-0105002 altered signaling downstream of NMDAR and thus functionally and structurally attenuating synaptic plasticity through respective regulation of the NR2B/GluR1/CaMKIIα and Rac1/RhoA pathways. These findings suggest that the novel PSD95-nNOS inhibitor PCC-0105002 is an effective agent for alleviating neuropathic pain, and that it produces fewer motor coordination-associated side effects than do NMDAR antagonists.

Salidroside alleviates diabetic neuropathic pain through regulation of the AMPK-NLRP3 inflammasome axis.

High glucose (HG)-induced nucleotide-binding and oligomerization (NACHT) domain, leucine-rich repeat (LRR), and pyrin domain (PYD)-containing protein 3 (NLRP3) inflammasome activation leads to diabetic neuropathic pain. We recently showed that salidroside could suppress NLRP3 inflammasome activation in hepatocytes exposed to HG. The aim of this study was to evaluate the analgesic effect of salidroside on diabetic rats and to explore its underlying mechanisms. Rat models with diabetic neuropathic pain were induced by high-fat diet feeding combined with low dose streptozotocin injections. Doses of salidroside at 50 and 100 mg.kg-1.day-1 were administered by gavage to diabetic rats for 6 weeks. Mechanical allodynia test, thermal hyperalgesia test and biochemical analysis were performed to evaluate therapeutic effects. Primary dorsal root ganglion (DRG) cells exposed to HG at 45 mM were used to further study the effects of salidroside on the AMP-activated protein kinase (AMPK)-NLRP3 inflammasome axis and insulin sensitivity in vitro. Salidroside administration improved hyperglycemia, ameliorated insulin resistance, and alleviated neuropathic pain in diabetic rats. Moreover, salidroside induced AMPK activation and suppressed NLRP3 inflammasome activation in the DRGs of diabetic rats. In addition, salidroside treatment relieved oxidative stress, improved insulin sensitivity and regulated the AMPK-NLRP3 inflammasome axis in HG-treated DRGs in vitro. Furthermore, AMPK inhibition in vivo or AMPK silencing in vitro abolished the beneficial effects of salidroside on diabetic neuropathic pain. Together, these results indicate that salidroside alleviates diabetic neuropathic pain through its regulation of the AMPK-NLRP3 inflammasome axis in DRGs.

Targeting aurora kinase B alleviates spinal microgliosis and neuropathic pain in a rat model of peripheral nerve injury.

Peripheral nerve injury elicits spinal microgliosis, contributing to neuropathic pain. The aurora kinases A (AURKA), B (AURKB), and C (AURKC) are potential therapeutic targets in proliferating cells. However, their role has not been clarified in microglia. The aim of this study was to examine the regulation of aurora kinases and their roles and druggability in spinal microgliosis and neuropathic pain. Sprague-Dawley rats received chronic constriction injury (CCI). Gene expression of aurora kinases A-C was evaluated by quantitative RT-PCR and western blot, respectively, in spinal cords at 1, 3, 7, and 14 days after CCI. AURKB gene and protein expression was up-regulated concomitantly with the development of spinal microgliosis and neuropathic pain. Using lentiviral over-expression and adeno-associated viral knockdown approaches, the function of AURKB was further investigated by western blot, immunohistochemistry, RNA sequencing, and pain behavior tests. We found that AURKB over-expression in naive rats caused spinal microgliosis and pain hypersensitivity, whereas AURKB knockdown reduced microgliosis and alleviated CCI-induced neuropathic pain. Accordingly, RNA sequencing data revealed down-regulation of genes critically involved in signaling pathways associated with spinal microgliosis and neuropathic pain after AURKB knockdown in CCI rats. To examine its therapeutic potential for treatment of neuropathic pain, animals were treated intrathecally with the pharmacological AURKB inhibitor AZD1152-HQPA resulting in the alleviation of CCI-induced pain. Taken together, our findings indicated that AURKB plays a critical role in spinal microgliosis and neuropathic pain. Targeting AURKB may be an efficient method for treatment of neuropathic pain subsequent to peripheral nerve injury.

CXCR3 contributes to neuropathic pain via ERK activation in the anterior cingulate cortex.

The anterior cingulate cortex (ACC) is activated by noxious stimuli and is involved in the affective component of pain processing; but its role in the sensory component of pain remains largely unknown. Studies have verified that Chemokine (C-X-C motif) receptor 3 (CXCR3) is involved in nociceptive sensitization in the spinal cord after peripheral nerve injury; however, the expression of CXCR3 in the ACC and its role in neuropathic pain has not been reported. Here, we showed that CXCR3 co-localized with neurons in the ACC and the upregulation of CXCR3 corresponded with hypersensitive behaviors after a chronic constriction injury of the sciatic nerve. Pharmacological blockade of CXCR3 using local injection of its inhibitor, AMG487, into the ACC significantly attenuated hyperalgesia induced by chronic constriction injury and suppressed the phosphorylation of extracellular signal-regulated kinase (ERK). Collectively, these results suggest that CXCR3 in the ACC is involved in hyperalgesia induced by peripheral nerve injury and ERK may be a downstream target.

The Role of Astrocytes in the Modulation ofK+-Cl--Cotransporter-2 Function.

Neuropathic pain is characterized by spontaneous pain, pain sensations, and tactile allodynia. The pain sensory system normally functions under a fine balance between excitation and inhibition. Neuropathic pain arises when this balance is lost for some reason. In past reports, various mechanisms of neuropathic pain development have been reported, one of which is the downregulation of K+-Cl--cotransporter-2 (KCC2) expression. In fact, various neuropathic pain models indicate a decrease in KCC2 expression. This decrease in KCC2 expression is often due to a brain-derived neurotrophic factor that is released from microglia. However, a similar reaction has been reported in astrocytes, and it is unclear whether astrocytes or microglia are more important. This review discusses the hypothesis that astrocytes have a crucial influence on the alteration of KCC2 expression.

Spinal Activation of Tropomyosin Receptor Kinase-B Recovers the Impaired Endogenous Analgesia in Neuropathic Pain Rats.

BACKGROUND: Although endogenous analgesia plays an important role in controlling pain states, chronic pain patients exhibit decreased endogenous analgesia compared to healthy individuals. In rats, noxious stimulus-induced analgesia (NSIA), which is an indicator of endogenous analgesia, diminished 6 weeks after spinal nerve ligation (SNL6W). A recent study in rats with deleted noradrenergic fibers demonstrated that the noradrenergic fibers were essential to NSIA. It has also been reported that brain-derived neurotrophic factor increased spinal noradrenergic fibers. Therefore, this study examined the effect of TrkB activation, which is the receptor for brain-derived neurotrophic factor, on impaired NSIA in SNL6W rats. In addition, we also examined the effect of endogenous analgesia on acute incisional pain. METHODS: After 5 daily intraperitoneal injections of 7,8-dihydroxyflavone (7,8-DHF, TrkB agonist, 5 mg/kg), NSIA was examined by measuring the withdrawal threshold increment in the left (contralateral to nerve ligation) hindpaw at 30 minutes after capsaicin injection (250 µg) in the forepaw. K252a (TrkB antagonist, 2 µg) was administrated intrathecally for 5 days. Idazoxan (α2 adrenoceptor antagonist, 30 µg), atropine (muscarinic antagonist, 30 µg), and propranolol (nonselective ß adrenoceptor antagonist, 30 µg) were administered intrathecally for 15 minutes before capsaicin injection. Microdialysis and immunohistochemistry were performed to examine the noradrenergic plasticity in the spinal dorsal horn. A hindpaw incision was performed on the left (contralateral to nerve ligation) hindpaw. Data were analyzed by 1-way analyses of variance or 2-way repeated-measures 1-way analysis of variance followed by a Student t test with Bonferroni correction. RESULTS: Five daily intraperitoneal injections of 7,8-DHF restored the attenuated NSIA in SNL6W rats (n = 7, P = .002; estimated treatment effect [95% CI]: 62.9 [27.0-98.7] g), with this effect blocked by 5 daily intrathecal coadministrations of K252a (n = 6, P < .001; -57.8 [-78.3 to -37.2] g). This effect was also inhibited by a single intrathecal administration of idazoxan (n = 8, P < .001; -61.6 [-92.4 to -30.9] g) and atropine (n = 8, P = .003; -52.6 [-73.3 to -31.9] g), but not by propranolol. Furthermore, 7,8-DHF increased the noradrenergic fiber in the spinal dorsal horn and the noradrenaline release in response to the capsaicin injection in the forepaw in SNL6W rats. In addition, repeated injections of 7,8-DHF prevented delayed recovery from incisional pain in SNL6W rats. CONCLUSIONS: Spinal activation of TrkB may recover the attenuated endogenous analgesia by improving the adrenergic plasticity, thereby leading to prevention of pain prolongation after surgery.

Inhibition of Fatty Acid Amide Hydrolase Improves Depressive-Like Behaviors Independent of Its Peripheral Antinociceptive Effects in a Rat Model of Neuropathic Pain.

BACKGROUND: Neuropathic pain is often associated with depression. Enhancing endocannabinoids by fatty acid amide hydrolase (FAAH) inhibitors relieves neuropathic pain and stress-induced depressive-like behaviors in animal models. However, it is unclear whether FAAH inhibitor can relieve neuropathic pain-induced depression by or not by its antinociceptive effects. METHODS: Adult male Wistar rats with chronic constriction injury (CCI) to the sciatic nerve were treated with the systemic FAAH inhibitor URB597 (5.8 mg·kg·day, intraperitoneally) or peripherally acting FAAH inhibitor URB937 (1.6 mg·kg·d, intraperitoneally; n = 11-12). The treatment was applied from the 15th day after surgery and continued for 15 days. Mechanical withdrawal threshold was examined by Von Frey test before surgery and on the 28th day after CCI. Depressive-like behaviors were evaluated by forced swimming test (FST) and novelty-suppressed feeding (NSF) after 15-day treatment. The levels of anandamide and 2-arachidonoylglycerol in hippocampus were examined by liquid chromatography and mass spectrometry. Hippocampal neurogenesis including proliferation, differentiation, and survival of newborn cells was assessed by immunohistochemistry. RESULTS: After CCI injury, the rats developed significantly nociceptive and depressive-like behaviors, indicated by persistent mechanical hypersensitivity in Von Frey test, significantly prolonged immobility time in FST (sham: 84.2 ± 13.4 seconds versus CCI: 137.9 ± 18.8 seconds; P < .001), and protracted latency to feed in NSF (sham: 133.4 ± 19.4 seconds versus CCI: 234.9 ± 33.5 seconds; P < .001). For the CCI rats receiving treatment, compared to vehicle placebo group, pain threshold was increased by both URB597 (3.1 ± 1.0 vs 11.2 ± 1.2 g; P < .001) and URB937 (3.1 ± 1.0 vs 12.1 ± 1.3 g; P < .001). Immobility time of FST was reduced by URB597 (135.8 ± 16.6 vs 85.3 ± 17.2 seconds; P < .001) but not by URB937 (135.8 ± 16.6 vs 129.6 ± 17.8 seconds; P = .78). Latency to feed in NSF was also reduced by URB597 (235.9 ± 30.5 vs 131.8 ± 19.8 seconds; P < .001) but not by URB937 (235.9 ± 30.5 vs 232.2 ± 33.2 seconds; P = .72). Meanwhile, CCI decreased the number of proliferating cells and reduced survival of new mature neurons in hippocampus. URB597 but not URB937 treatment improved these cellular deficits. CONCLUSIONS: Inhibition of FAAH can improve depressive-like behaviors induced by neuropathic pain independent of its peripheral antinociceptive action. Enhanced neurogenesis in hippocampus might contribute to the antidepressive effects of URB597.

The Involvement of DDAH1 in the Activation of Spinal NOS Signaling in Early Stage of Mechanical Allodynia Induced by Exposure to Ischemic Stress in Mice.

The pathophysiological mechanism of central post-stroke pain (CPSP) is complicated and not well understood. Recently, it has been reported that an increase in the levels of spinal nitric oxide synthetase (NOS) occurs in cerebral ischemia, and spinal NOS is involved in the development of neuropathic pain. The aim of this study was to elucidate the mechanism of spinal NOS signaling in the development of CPSP. Male ddY mice were subjected to 30-min long bilateral carotid artery occlusion (BCAO). The withdrawal responses to mechanical stimuli were significantly increased as determined with von Frey test on days 1 and 3 after BCAO. Protein expression of spinal N(G),N(G)-dimethylarginine dimethylaminohydralase 1 (DDAH1), a key enzyme involved in the metabolism of the endogenous NOS, increased on day 1 after BCAO, but not on day 3. Intrathecal (i.t.) injection of PD404182, a DDAH1 inhibitor, significantly suppressed mechanical allodynia on day 1, but not on day 3 after BCAO. In addition, i.t. administration of NG-nitro-L-arginine methyl ester (L-NAME), a non-selective NOS inhibitor, significantly blocked mechanical allodynia on days 1 and 3 after BCAO. Furthermore, BCAO-induced increment of spinal NOS activity was inhibited by the pretreatment with PD404182. These results suggest that mechanical allodynia in the early stage of CPSP is caused by increment of NOS activity through upregulated DDAH1 in the spinal cord.

Transcription-independent expression of PKMζ in the anterior cingulate cortex contributes to chronically maintained neuropathic pain.

Protein kinase M ζ is well known for its role in maintaining memory and pain. Previously, we revealed that the activation of protein kinase M ζ in the anterior cingulate cortex plays a role in sustaining neuropathic pain. However, the mechanism by which protein kinase M ζ is expressed in the anterior cingulate cortex by peripheral nerve injury, and whether blocking of protein kinase M ζ using its inhibitor, zeta inhibitory peptide, produces analgesic effects in neuropathic pain maintained chronically after injury, have not previously been resolved. In this study, we show that protein kinase M ζ expression in the anterior cingulate cortex is enhanced by peripheral nerve injury in a transcription-independent manner. We also reveal that the inhibition of protein kinase M ζ through zeta inhibitory peptide treatment is enough to reduce mechanical allodynia responses in mice with one-month-old nerve injuries. However, the zeta inhibitory peptide treatment was only effective for a limited time.

Spinal CXCL9 and CXCL11 are not involved in neuropathic pain despite an upregulation in the spinal cord following spinal nerve injury.

Chemokines-mediated neuroinflammation in the spinal cord plays a critical role in the pathogenesis of neuropathic pain. Chemokine CXCL9, CXCL10, and CXCL11 have been identified as a same subfamily chemokine which bind to CXC chemokine receptor 3 to exert functions. Our recent work found that CXCL10 is upregulated in spinal astrocytes after spinal nerve ligation (SNL) and acts on chemokine receptor CXCR3 on neurons to contribute to central sensitization and neuropathic pain, but less is known about CXCL9 and CXCL11 in the maintenance of neuropathic pain. Here, we report that CXCL9 and CXCL11, same as CXCL10, were increased in spinal astrocytes after SNL. Surprisingly, inhibition of CXCL9 or CXCL11 by spinal injection of shRNA lentivirus did not attenuate SNL-induced neuropathic pain. In addition, intrathecal injection of CXCL9 and CXCL11 did not produce hyperalgesia or allodynia behaviors, and neither of them induced ERK activation, a marker of central sensitization. Whole-cell patch clamp recording on spinal neurons showed that CXCL9 and CXCL11 enhanced both excitatory synaptic transmission and inhibitory synaptic transmission, whereas CXCL10 only produced an increase in excitatory synaptic transmission. These results suggest that, although the expression of CXCL9 and CXCL11 are increased after SNL, they may not contribute to the maintenance of neuropathic pain.

Histone deacetylase inhibition inhibits brachial plexus avulsion-induced neuropathic pain.

INTRODUCTION: Neuropathic pain induced by brachial plexus avulsion (BPA) is a pathological condition. We hypothesized that inhibition of histone deacetylase (HDAC) could suppress BPA-induced neuropathic pain through inhibition of transient reception potential (TRP) overexpression and protein kinase B (Akt)-mediated mammalian target of rapamycin (mTOR) activation. METHODS: We generated a rat BPA model; administered HDAC inhibitor tricostatin A (TSA) for 7 days postsurgery; and assessed the effects on HDAC expression, Akt phosphorylation, neuroinflammation, and mTOR activation. RESULTS: TSA treatment alleviated BPA-induced mechanical hyperalgesia, suppressed Akt phosphorylation, and increased HDAC. We found suppressed proinflammatory cytokine levels, TRPV1 and TRPM8 expression, and mTOR activity in TSA-treated BPA rats. DISCUSSION: Our results suggest that altered HDAC and Akt signaling are involved in BPA-induced neuropathic pain and that inhibition of HDAC could be an effective therapeutic approach in reducing neuropathic pain. Muscle Nerve 58: 434-440, 2018.

PKM2 is involved in neuropathic pain by regulating ERK and STAT3 activation in rat spinal cord.

BACKGROUND: Pyruvate kinase isozymes M2 (PKM2), as a member of pyruvate kinase family, plays a role of glycolytic enzyme in glucose metabolism. It also functions as protein kinase in cell proliferation, signaling, immunity, and gene transcription. In this study, the role of PKM2 in neuropathic pain induced by chronic constriction injury (CCI) was investigated. METHODS: Rats were randomly grouped to establish CCI models. PKM2, extracellular regulated protein kinases (EKR), p-ERK, signal transducers and activators of transcription (STAT3), p-STAT3, phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and p-PI3K/AKT proteins expression in spinal cord was examined by Western blot analysis. Cellular location of PKM2 was examined by immunofluorescence. Knockdown of PKM2 was achieved by intrathecal injection of specific small interfering RNA (siRNA). Von Frey filaments and radiant heat tests were performed to determine mechanical allodynia and thermal hyperalgesia respectively. Lactate and adenosine triphosphate (ATP) contents were measured by specific kits. Tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) levels were detected by ELISA kits. RESULTS: CCI markedly increased PKM2 level in rat spinal cord. Double immunofluorescent staining showed that PKM2 co-localized with neuron, astrocyte, and microglia. Intrathecal injection of PKM2 siRNA not only attenuated CCI-induced ERK and STAT3 activation, but also attenuated mechanical allodynia and thermal hyperalgesia induced by CCI. However, PKM2 siRNA failed to inhibit the activation of AKT. In addition, PKM2 siRNA significantly suppressed the production of lactate and pro-inflammatory mediators. CONCLUSION: Our findings demonstrate that inhibiting PKM2 expression effectively attenuates CCI-induced neuropathic pain and inflammatory responses in rats, possibly through regulating ERK and STAT3 signaling pathway.

Pain vulnerability and DNA methyltransferase 3a involved in the affective dimension of chronic pain.

Chronic pain with comorbid emotional disorders is a prevalent neurological disease in patients under various pathological conditions, yet patients show considerable difference in their vulnerability to developing chronic pain. Understanding the neurobiological basis underlying this pain vulnerability is essential to develop targeted therapies of higher efficiency in pain treatment of precision medicine. However, this pain vulnerability has not been addressed in preclinical pain research in animals to date. In this study, we investigated individual variance in both sensory and affective/emotional dimensions of pain behaviors in response to chronic neuropathic pain condition in a mouse model of chronic pain. We found that mice displayed considerably diverse sensitivities in the chronic pain-induced anxiety- and depression-like behaviors of affective pain. Importantly, the mouse group that was more vulnerable to developing anxiety was also more vulnerable to developing depressive behavior under the chronic pain condition. In contrast, there was relatively much less variance in individual responses in the sensory dimension of pain sensitization. Molecular analysis revealed that those mice vulnerable to developing the emotional disorders showed a significant reduction in the protein level of DNA methyltransferase 3a in the emotion-processing central nucleus of the amygdala. In addition, social stress also revealed significant individual variance in anxiety behavior in mice. These findings suggest that individual pain vulnerability may be inherent mostly in the emotional/affective component of chronic pain and remain consistent in different aspects of negative emotion, in which adaptive changes in the function of DNA methyltransferase 3a for DNA methylation in central amygdala may play an important role. This may open a new avenue of basic research into the neurobiological mechanisms underlying pain vulnerability.

Selective suppression of the JNK-MMP2/9 signal pathway by tetramethylpyrazine attenuates neuropathic pain in rats.

BACKGROUND: Activated astrocytes release matrix metalloproteinase-2/9 (MMP-2/9) to induce central sensitization and maintain neuropathic pain. However, the mechanisms involved in the activation of MMP-2/9 on astrocytes during pain remain poorly understood. Meanwhile, there is a lack of effective treatment to inhibit the activation of MMP-2/9 on astrocytes. In this study, we aim to investigate the effect of tetramethylpyrazine (TMP), a natural compound with analgesic effects but unknown mechanisms, on MMP-2/9 in neuropathic pain. METHODS: The nociception was assessed by measuring the incidence of foot withdrawal in response to mechanical indentation in rats (n = 6). Cell signaling was assayed using western blotting (n = 6) and immunohistochemistry (n = 5). The astrocyte cell line C8-D1A was cultured to investigate the in vitro effects. RESULTS: TMP significantly attenuated the maintenance of chronic constrictive injury (CCI)-induced neuropathic pain, inhibited the activation of astrocytes, and decreased the expression of MMP-2/9. Furthermore, our results indicated that TMP could selectively suppress JNK activity but had no notable effects on ERK and p38. Our study also revealed that the effect of TMP may be dependent on the inhibition of TAK1. CONCLUSIONS: Inhibition of astrocyte activation in the spinal cord by tetramethylpyrazine may have utility in the treatment of CCI-induced neuroinflammation, and our results further implicate JNK-MMP-2/9 as a novel target for the attenuation of neuropathic pain.

Prophylactic inhibition of neutrophil elastase prevents the development of chronic neuropathic pain in osteoarthritic mice.

BACKGROUND: A subset of osteoarthritis (OA) patients experience joint pain with neuropathic characteristics. Mediators such as neutrophil elastase, a serine proteinase, may be released during acute OA inflammatory flares. We have previously shown that local administration of neutrophil elastase causes joint inflammation and pain via activation of proteinase-activated receptor-2 (PAR2). The aim of this study was to examine the contribution of endogenous neutrophil elastase and PAR2 to the development of joint inflammation, pain, and neuropathy associated with monoiodoacetate (MIA)-induced experimental OA. METHODS: MIA (0.3 mg/10 µl) was injected into the right knee joint of male C57BL/6 mice (20-34 g). Joint inflammation (edema, leukocyte kinetics), neutrophil elastase proteolytic activity, tactile allodynia, and saphenous nerve demyelination were assessed over 14 days post-injection. The effects of inhibiting neutrophil elastase during the early inflammatory phase of MIA (days 0 to 3) were determined using sivelestat (50 mg/kg i.p.) and serpinA1 (10 µg i.p.). Involvement of PAR2 in the development of MIA-induced joint inflammation and pain was studied using the PAR2 antagonist GB83 (5 µg i.p. days 0 to 1) and PAR2 knockout animals. RESULTS: MIA caused an increase in neutrophil elastase proteolytic activity on day 1 (P < 0.0001), but not on day 14. MIA also generated a transient inflammatory response which peaked on day 1 (P < 0.01) then subsided over the 2-week time course. Joint pain appeared on day 1 and persisted to day 14 (P < 0.0001). By day 14, the saphenous nerve showed signs of demyelination. Early treatment with sivelestat and serpinA1 blocked the proteolytic activity of neutrophil elastase on day 1 (P < 0.001), and caused lasting improvements in joint inflammation, pain, and saphenous nerve damage (P < 0.05). MIA-induced synovitis was reversed by early treatment with GB83 and attenuated in PAR2 knockout mice (P < 0.05). PAR2 knockout mice also showed reduced MIA-induced joint pain (P < 0.0001) and less nerve demyelination (P = 0.81 compared to saline control). CONCLUSIONS: Neutrophil elastase and PAR2 contribute significantly to the development of joint inflammation, pain, and peripheral neuropathy associated with experimental OA, suggesting their potential as therapeutic targets.

Cellular and Molecular Mechanisms of Calcium/Calmodulin-Dependent Protein Kinase II in Chronic Pain.

Chronic pain, often defined as any pain lasting more than 3 months, is poorly managed because of its multifaceted and complex mechanisms. Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine kinase that plays a fundamental role in synaptic plasticity, learning, and memory. Recent emerging evidence demonstrates increased expression and activity of CaMKII in the spinal cord and dorsal root ganglia of various chronic pain models. Moreover, our previous studies also find that inhibiting CaMKII could attenuate inflammatory pain and neuropathic pain. In this review, we provide evidence for the involvement of CaMKII in the initiation and development of chronic pain, including neuropathic pain, bone cancer pain, and inflammatory pain. Novel CaMKII inhibitors with potent inhibitory effect and high specificity may be alternative therapeutic strategies for the management of chronic pain in the future.

Upregulation of neuronal kynurenine 3-monooxygenase mediates depression-like behavior in a mouse model of neuropathic pain.

Pain and depression often co-occur, but the underlying mechanisms have not been elucidated. Here, we used the spared nerve injury (SNI) model in mice to induce both neuropathic pain and depression-like behavior. We investigated whether brain interleukin (IL)-1 signaling and activity of kynurenine 3-monoxygenase (KMO), a key enzyme for metabolism of kynurenine into the neurotoxic NMDA receptor agonist quinolinic acid, are necessary for comorbid neuropathic pain and depression-like behavior. SNI mice showed increased expression levels of Il1b and Kmo mRNA in the contralateral side of the brain. The SNI-induced increase of Kmo mRNA was associated with increased KMO protein and elevated quinolinic acid and reduced kynurenic acid in the contralateral hippocampus. The increase in KMO-protein in response to SNI mostly took place in hippocampal NeuN-positive neurons rather than microglia. Inhibition of brain IL-1 signaling by intracerebroventricular administration of IL-1 receptor antagonist after SNI prevented the increase in Kmo mRNA and depression-like behavior measured by forced swim test. However, inhibition of brain IL-1 signaling has no effect on mechanical allodynia. In addition, intracerebroventricular administration of the KMO inhibitor Ro 61-8048 abrogated depression-like behavior without affecting mechanical allodynia after SNI. We show for the first time that the development of depression-like behavior in the SNI model requires brain IL-1 signaling and activation of neuronal KMO, while pain is independent of this pathway. Inhibition of KMO may represent a promising target for treating depression.

Down-Regulation of miRNA-128 Contributes to Neuropathic Pain Following Spinal Cord Injury via Activation of P38.

BACKGROUND Neuropathic pain (NPP) arises from a lesion or dysfunction of the somatosensory nervous system. Recent studies have demonstrated multiple microRNAs (miRNAs) play key roles in NPP development. This study aimed to investigate the effects of miR-128 on microglial cells. MATERIAL AND METHODS We established a compressive spinal cord injury (SCI) model and collected the spinal cord segment-derived conditioned medium (CM). We then measured the expression of miR-128 in the murine microglial cell line BV2 treated with CM-SCI or CM obtained from control (CM-NC). Furthermore, lentivirus production of miR-128 and scrambled control were transfected into BV2 cells, which were first treated with CM-SCI or CM-NC. Moreover, the effects of miR-128 on cell viability, M1/M2 microglial gene expression, inflammatory cytokines concentration, and the protein expression of P38 and phosphorylated P38 (P-P38) were investigated. RESULTS The expression of miR-128 was downregulated in murine microglial BV2 cells treated with CM-SCI. Overexpression of miR-128 markedly promoted the viability of murine microglial cells. In addition, miR-128 overexpression significantly decreased the expression levels of microglial M1 phenotypic markers CD86 and CD32, and increased the expression levels of M2 phenotypic markers Arg1 and CD206. Furthermore, miR-128 overexpression obviously decreased the concentration of TNF-α, IL-1ß, and IL-6. We found that miR-128 overexpression significantly downregulated the expression levels of P38 andP-P38. CONCLUSIONS Our findings indicate that down-regulation of miR-128 in murine microglial cells may contribute to the development of NPP following SCI via activation of P38. MiR-128 may be a potential intervention target for NPP.