Evidence that gabapentin reduces neuropathic pain by inhibiting the spinal release of glutamate

Gabapentin is an anticonvulsant that successfully treats many neuropathic pain syndromes, although the mechanism of its antihyperalgesic action remains elusive. This study aims to help delineate gabapentin's antihyperalgesic mechanisms. We assessed the effectiveness of gabapentin at decreasing mechanical and cold hypersensitivity induced in a rat model of neuropathic pain. Thus, we compared the effectiveness of pre‐ or post‐treatment with systemic or intrathecal (i.t.) gabapentin at reducing the development and maintenance of the neuropathic pain symptoms. Gabapentin successfully decreased mechanical and cold hypersensitivity, both as a pretreatment and post‐treatment. Furthermore, both i.t. and systemic administration of gabapentin were effective in reducing the behavioral hypersensitivity; however, the i.t. administration was superior to the systemic. We also examined gabapentin's effects at inhibiting hindpaw formalin‐induced release of excitatory amino acids (EAAs) in the spinal cord dorsal horn (SCDH) both in naïve rats and in rats with neuropathic pain. We present the first evidence that gabapentin reduces the formalin‐induced release of both glutamate and aspartate in SCDH. Furthermore, i.t. gabapentin reduces the enhanced noxious stimulus‐induced spinal release of glutamate seen in neuropathic rats. These data suggest that gabapentin reduces neuropathic pain symptoms by inhibiting the release of glutamate in the SCDH.     

Estrogen alleviates neuropathic pain induced after spinal cord injury by inhibiting microglia and astrocyte activation

Neuropathic pain after spinal cord injury (SCI) is developed in about 80% of SCI patients and there is no efficient therapeutic drug to alleviate SCI-induced neuropathic pain. Here we examined the effect of estrogen on SCI-induced neuropathic pain at below-level and its effect on neuroinflammation as underlying mechanisms. Neuropathic pain is developed at late phase after SCI and a single dose of 17β-estradiol (100, 300?μg/kg) were administered to rats with neuropathic pain after SCI through intravenous injection. As results, both mechanical allodynia and thermal hyperalgesia were significantly reduced by 17β-estradiol compared to vehicle control. Both microglia and astrocyte activation in the lamina I and II of L4-5 dorsal horn was also inhibited by 17β-estradiol. In addition, the levels of p-p38MAPK and p-ERK known to be activated in microglia and p-JNK known to be activated in astrocyte were significantly decreased by 17β-estradiol. Furthermore, the mRNA expression of inflammatory mediators such as Il-1β, Il-6, iNos, and Cox-2 was more attenuated in 17β-estradiol-treated group than in vehicle-treated group. Particularly, we found that the analgesic effect by 17β-estradiol was mediated via estrogen receptors, which are expressed in dorsal horn neurons. These results suggest that 17β-estradiol may attenuate SCI-induced neuropathic pain by inhibiting microglia and astrocyte activation followed inflammation.     

Intrathecal lidocaine pretreatment attenuates immediate neuropathic pain by modulating Nav1.3 expression and decreasing spinal microglial activation

Background  

Intrathecal lidocaine reverses tactile allodynia after nerve injury, but whether neuropathic pain is attenuated by intrathecal lidocaine pretreatment is uncertain.     

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.     

Zingiber officinale Roscoe rhizome extract alleviates neuropathic pain by inhibiting neuroinflammation in mice

BackgroundCurrent therapies for neuropathic pain are generally symptomatic and possess several side effects, limiting their prolonged usage.Hypothesis/PurposeThus, it is urgent to develop novel and safe candidates for the management of this chronical condition. For this purpose, we investigated the analgesic effect of a standardized extract from Zingiber officinale Roscoe rhizomes (ZOE) obtained by CO₂ supercritical extraction, in a mice model of peripheral neuropathy. We also explored the mechanism of action of ZOE and its main constituents using an in vitro model of neuroinflammation.MethodsPeripheral mono-neuropathy was induced in mice, by spared nerve injury (SNI). The analgesic effect of ZOE after oral administration was assessed by measuring mechanical and thermal allodynia in SNI mice. The mechanism of action of ZOE and its main constituents were investigated using spinal cords samples and in an in vitro model of neuroinflammation by ELISA, western blotting and immunofluorescence techniques.ResultsOral administration of ZOE 200 mg kg⁻¹ ameliorated mechanical and thermal allodynia in SNI mice, with a rapid and a long-lasting effect. ZOE did not alter locomotor activity. In BV2 cells and spinal cord samples, ZOE, 6-gingerol and 6-shogaol reduced pERK levels, whereas ZOE and terpene fraction reduced HDAC1 protein levels, inhibited NF-κB signalling activation and decreased IL-1β, TNF-α and IL-6 release. ZOE and each tested constituent had a positive effect on inflammation-impaired SH-SY5Y cell viability.ConclusionsThe oral administration of ZOE attenuated SNI-induced neuropathic pain symptoms by reducing spinal neuroinflammation, suggesting ZOE as a novel and interesting candidate for the management of neuropathic pain.     

GCH1-regulated miRNAs are potential targets for microglial activation in neuropathic pain

Neuropathic pain (NP) is a chronic pain directly caused by injury or disease of the somatosensory nervous system. Previous studies suggest that GTP cyclohydrolase I (GCH1) may play a pivotal role in microglial activation, which has been shown to be essential for NP. However, its underlying mechanisms in microglial activation remain unclear. A wide range of microRNAs (miRNAs) have been found to be involved in microglial activation-induced NP. To identify the miRNAs regulated by GCH1 and predict their functions in the progression of microglial activation, we analyzed the miRNA expression profiles of GCH1-knockdown (KD) BV2 microglial cells. Small RNA-sequencing analysis revealed 13 differentially expressed (DE) miRNAs in GCH1-KD cells. The target genes of DE miRNAs mainly participate in PI3K-Akt signaling pathway, peroxisome and ferroptosis. The miRNA–mRNA regulatory network analysis showed that GCH1, MAP4K5 and YWHAB acted as hub genes. qRT-PCR results further verified the expression levels of mmu-miR-1a-3p, mmu-miR-133a-3p, mmu-miR-7a-5p and mmu-miR-10a-5p in GCH1-KD cells, which were consistent with the sequencing data. In addition, our data indicated that overexpression of mmu-miR-133a-3p alleviated the pro-inflammatory cytokines IL-1β and IL-6 production induced by lipopolysaccharide (LPS), indicating that mmu-miR-133a-3p has a negative effect on microglial activation. Taken together, our findings suggest that many miRNAs regulated by GCH1 may be involved in microglial activation, which may provide new potential targets for GCH1 in the pathogenesis of NP.     

Carbon monoxide dehydrogenase in mycobacteria possesses a nitric oxide dehydrogenase activity

CO dehydrogenase (CO-DH) catalyzes the oxidation of CO to CO(2) in carboxydobacteria. Cell-free extracts prepared from several mycobacteria, including Mycobacterium tuberculosis H37Ra, showed NO dehydrogenase (NO-DH) activity in a reaction mixture containing sodium nitroprusside (SNP) as the source of NO. The association of the NO-DH activity with CO-DH was revealed by activity staining and confirmed by enzyme assay with purified CO-DH from Mycobacterium sp. strain JC1, a carboxydotrophic mycobacterium. SNP stimulated the production of CO-DH with a coincidental increase in NO-DH activity in the bacterium, further supporting this association and implying the existence of a possible SNP-induced CO-DH gene expression. The addition of purified CO-DH to cultures of Escherichia coli revealed that the enzyme protected E. coli from SNP-induced killing in a dose-dependant way. The present results indicate that mycobacterial CO-DH also acts as a NO-DH, which may function in the protection of mycobacterial pathogens from nitrosative stress during infection.     

Cannabinoid-mediated modulation of neuropathic pain and microglial accumulation in a model of murine type I diabetic peripheral neuropathic pain

Background

Despite the frequency of diabetes mellitus and its relationship to diabetic peripheral neuropathy (DPN) and neuropathic pain (NeP), our understanding of underlying mechanisms leading to chronic pain in diabetes remains poor. Recent evidence has demonstated a prominent role of microglial cells in neuropathic pain states. One potential therapeutic option gaining clinical acceptance is the cannabinoids, for which cannabinoid receptors (CB) are expressed on neurons and microglia. We studied the accumulation and activation of spinal and thalamic microglia in streptozotocin (STZ)-diabetic CD1 mice and the impact of cannabinoid receptor agonism/antagonism during the development of a chronic NeP state. We provided either intranasal or intraperitoneal cannabinoid agonists/antagonists at multiple doses both at the initiation of diabetes as well as after establishment of diabetes and its related NeP state.

Results

Tactile allodynia and thermal hypersensitivity were observed over 8 months in diabetic mice without intervention. Microglial density increases were seen in the dorsal spinal cord and in thalamic nuclei and were accompanied by elevation of phosphorylated p38 MAPK, a marker of microglial activation. When initiated coincidentally with diabetes, moderate-high doses of intranasal cannabidiol (cannaboid receptor 2 agonist) and intraperitoneal cannabidiol attenuated the development of an NeP state, even after their discontinuation and without modification of the diabetic state. Cannabidiol was also associated with restriction in elevation of microglial density in the dorsal spinal cord and elevation in phosphorylated p38 MAPK. When initiated in an established DPN NeP state, both CB1 and CB2 agonists demonstrated an antinociceptive effect until their discontinuation. There were no pronociceptive effects demonstated for either CB1 or CB2 antagonists.

Conclusions

The prevention of microglial accumulation and activation in the dorsal spinal cord was associated with limited development of a neuropathic pain state. Cannabinoids demonstrated antinociceptive effects in this mouse model of DPN. These results suggest that such interventions may also benefit humans with DPN, and their early introduction may also modify the development of the NeP state.     

Up-regulation of microglial CD11b expression by nitric oxide

Increased expression of CD11b, the beta-integrin marker of microglia, represents microglial activation during neurodegenerative inflammation. However, the molecular mechanism behind increased microglial CD11b expression is poorly understood. The present study was undertaken to explore the role of nitric oxide (NO) in the expression of CD11b in microglial cells. Bacterial lipopolysaccharide (LPS) induced the production of NO and increased the expression of CD11b in mouse BV-2 microglial cells and primary microglia. Either a scavenger of NO (PTIO) or an inhibitor of inducible nitric-oxide synthase (L-NIL) blocked this increase in microglial CD11b expression. Furthermore, co-microinjection of PTIO with LPS was also able to suppress LPS-mediated expression of CD11b and loss of dopaminergic neuronal fibers and neurotransmitters in striatum in vivo. Similarly, other inducers of NO production such as interferon-gamma, interleukin-1beta, human immunodeficiency virus type-1 gp120, and double-stranded RNA (poly(IC)) also increased the expression of CD11b in microglia through NO. The role of NO in the expression of CD11b was corroborated further by the expression of microglial CD11b by GSNO, an NO donor. Because NO transduces many intracellular signals via guanylate cyclase (GC), we investigated the role of GC, cyclic GMP (cGMP), and cGMP-activated protein kinase (PKG) in microglial expression of CD11b. Inhibition of LPS- and GSNO-mediated up-regulation of CD11b either by NS2028 (a specific inhibitor of GC) or by KT5823 and Rp-8-bromo-cGMP (specific inhibitors of PKG), and increase in CD11b expression either by 8-bromo-cGMP or by MY-5445 (a specific inhibitor of cGMP phosphodiesterase) alone suggest that NO increases microglial expression of CD11b via GC-cGMP-PKG. In addition, GSNO induced the activation of cAMP response element-binding protein (CREB) via PKG that was involved in the up-regulation of CD11b. This study illustrates a novel biological role of NO in regulating the expression of CD11b in microglia through GC-cGMP-PKG-CREB pathway that may participate in the pathogenesis of devastating neurodegenerative disorders.     

Attractylone attenuates sepsis-associated encephalopathy and cognitive dysfunction by inhibiting microglial activation and neuroinflammation

Multiple studies demonstrated that sepsis is a life-threatening state of organ dysfunction caused by infection and can induce neuroinflammation and cognitive impairment. The aim of this study was to evaluate the protective effects of attractylone (Atr) on sepsis-associated encephalopathy (SAE) and cognitive dysfunction. Moreover, we studied the underlying molecular mechanisms. We used an LPS-induced sepsis mouse model and evaluated the cognitive function with the Morris water maze and open field test. Neuronal damage in the hippocampus was assessed by immunohistochemical analysis. BV2 cells were used to identify the protective mechanism of Atr. The result showed that Atr attenuated LPS-induced cognitive impairment, neural apoptosis, inflammatory factors, and microglial activation. The in vitro experiment showed that Atr promoted silent information regulator 1 (SIRT1) expression and suppressed NFκB expression. Downregulation of SIRT1 reversed the protective effect of Atr in the LPS condition. Moreover, Atr-induced SIRT1 expression promoted BV2 from LPS-induced M1 to M2 phenotype. Taken together, these results indicated that Atr was a potential therapeutic agent for SAE and cognitive dysfunction.     

The citrus flavonone naringenin reduces lipopolysaccharide-induced inflammatory pain and leukocyte recruitment by inhibiting NF-κB activation

Lipopolysaccharide (LPS) is the major structural component of Gram-negative bacteria cell wall and a highly pro-inflammatory toxin. Naringenin is found in Citrus fruits and exhibits antioxidant and anti-inflammatory properties through inhibition of NF-κB activation but its effects in LPS-induced inflammatory pain and leukocyte recruitment were not investigated yet. We investigated the effects of naringenin in mechanical hyperalgesia, thermal hyperalgesia and leukocyte recruitment induced by intraplantar injection of LPS in mice. We found that naringenin reduced hyperalgesia to mechanical and thermal stimuli, myeloperoxidase (MPO, a neutrophil and macrophage marker) and N-acetyl-β-D-glucosaminidase (NAG, a macrophage marker) activities, oxidative stress and cytokine (TNF-α, IL-1β, IL-6, and IL-12) production in the paw skin. In the peritoneal cavity, naringenin reduced neutrophil and mononuclear cell recruitment, and abrogated MPO and NAG activity, cytokine and superoxide anion production, and lipid peroxidation. In vitro, pre-treatment with naringenin inhibited superoxide anion and cytokine (TNF-α, IL-1β, IL-6, and IL-12) production by LPS-stimulated RAW 264.7 macrophages. Finally, we demonstrated that naringenin inhibited NF-κB activation in vitro and in vivo. Therefore, naringenin is a promising compound to treat LPS-induced inflammatory pain and leukocyte recruitment.     

N-type voltage-dependent Ca channel in non-excitable microglial cells in mice is involved in the pathophysiology of neuropathic pain

Peripheral nerve injury induces neuropathic pain which is characterized by tactile allodynia and thermal hyperalgesia. N-type voltage-dependent Ca²⁺ channel (VDCC) plays pivotal roles in the development of neuropathic pain, since mice lacking Ca_v2.2, the pore-forming subunit of N-type VDCC, show greatly reduced symptoms of both tactile allodynia and thermal hyperalgesia. Our study on gene expression profiles of the Ca_v2.2 knockout (KO) spinal cord after spinal nerve ligation (SNL)-injury revealed altered expression of genes known to be expressed in microglia, raising an odd idea that N-type VDCC may function in not only excitable (neurons) but also non-excitable (microglia) cells in neuropathic pain state. In the present study, we have tested this idea by using a transgenic mouse line, in which suppression of Ca_v2.2 expression can be achieved specifically in microglia/macrophage by the application of tamoxifen. We found SNL-operated transgenic mice exhibited greatly reduced signs of tactile allodynia, whereas the degree of thermal hyperalgesia was almost the same as that of control. Immunohistochemical analysis of the transgenic lumbar spinal cord revealed reduced accumulation of Iba1-positive cells (microglia/macrophage) around the injured neurons, indicating microglial N-type VDCC is important for accumulation of microglia at the lesion sites. Although the mechanism of its activation is not clear at present, activation of N-type VDCC expressed in non-excitable microglial cells contributes to the pathophysiology of neuropathic pain.     

Oltipraz inhibits inducible nitric oxide synthase in vitro and inhibits nitric oxide production in activated microglial cells

The clinically relevant drug oltipraz (OPZ) has previously been shown to inhibit cytochrome P450 enzymes [Chem. Res. Toxicol. 13 (2000) 245]. The current study reveals that OPZ is also able to inhibit *NO formation by purified inducible nitric oxide synthase (iNOS) but not by neuronal nitric oxide synthase in hemoglobin assays. The inhibition of iNOS by OPZ is reversible and competitive with an IC(50) of 5.9 microM and Ki of 0.6 microM. In murine BV-2 microglial cells, an immortalized cell line that produces *NO in response to lipopolysaccharide (LPS), OPZ is able to block the formation of nitrite in LPS treated cells. The inhibitory effect of OPZ on LPS treated cells is not due to cell toxicity. Finally, treatment of cells with OPZ does not induce or suppress expression of iNOS protein as shown by Western blot analysis.     

Optogenetic activation of spinal microglia triggers chronic pain in mice

Spinal microglia are highly responsive to peripheral nerve injury and are known to be a key player in pain. However, there has not been direct evidence showing that selective microglial activation in vivo is sufficient to induce chronic pain. Here, we used optogenetic approaches in microglia to address this question employing CX3CR1^creER/+: R26^LSL-ReaChR/+ transgenic mice, in which red-activated channelrhodopsin (ReaChR) is inducibly and specifically expressed in microglia. We found that activation of ReaChR by red light in spinal microglia evoked reliable inward currents and membrane depolarization. In vivo optogenetic activation of microglial ReaChR in the spinal cord triggered chronic pain hypersensitivity in both male and female mice. In addition, activation of microglial ReaChR up-regulated neuronal c-Fos expression and enhanced C-fiber responses. Mechanistically, ReaChR activation led to a reactive microglial phenotype with increased interleukin (IL)-1β production, which is likely mediated by inflammasome activation and calcium elevation. IL-1 receptor antagonist (IL-1ra) was able to reverse the pain hypersensitivity and neuronal hyperactivity induced by microglial ReaChR activation. Therefore, our work demonstrates that optogenetic activation of spinal microglia is sufficient to trigger chronic pain phenotypes by increasing neuronal activity via IL-1 signaling.

This study uses red light activation of channelrhodopsin in spinal microglia to trigger chronic pain hypersensitivity in awake mice, revealing that optogenetic activation of microglia increases IL-1β production via inflammasome activation and calcium elevation, leading to neuronal hyperactivity and chronic pain.