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Serpina3n is upregulated in mouse lumbar DRGs after spared nerve injury, a model of neuropathic pain. (a) Real-time qPCR analysis of Serpina3n expression in L3–L5 DRGs after SNI,with PPIA serving as reference gene (n = 4 mice per time point; *P < 0.05 compared to sham DRG; one-way ANOVA, Tukey's post hoc test). (b) Western blot analysis and quantification of SerpinA3N expression (55-kDa band) in L3–L4 DRGs after SNI normalized to α-tubulin expression (n = 3–5 mice per time point, one-way ANOVA, Tukey's post hoc test). (c) SerpinA3-like immunoreactivity in peptidergic (CGRP) and non-peptidergic (IB4-binding) nociceptors and large-diameter neurons (NF200) in L3–L4 DRG sections from naive mice (arrows indicate areas of colocalization). (d) Quantitative analysis of SerpinA3-like immunoreactivity in L3–L4 DRGs 3 d after SNI as compared to sham treatment (n = at least 3 DRG sections/mouse, 3 mice per treatment group; *P < 0.05 compared to sham; two-tailed unpaired t-test) and examples of upregulation in the CGRP-expressing population. (e) SerpinA3 and ATF3 staining in L3–L4 DRGs at day 1 after SNI showing SerpinA3+/ATF3+ (white arrows), Serpina3+/ATF3− (white arrowheads) and SerpinA3−/ATF3+ (yellow arrowheads) neurons. (f) Western blot analysis of SerpinA3N expression in lysates and medium from cultured DRG neurons. (g) Western blot analysis and quantification of SerpinA3N expression in lumbar spinal cord after SNI (n = 3–5 mice per time point, *P < 0.05 compared to sham, one-way ANOVA, Tukey's post hoc test). Scale bars, 100 μm (c–e). Error bars are means ± s.e.m.
Source publication
Neuropathic pain is a major, intractable clinical problem and its pathophysiology is not well understood. Although recent gene expression profiling studies have enabled the identification of novel targets for pain therapy, classical study designs provide unclear results owing to the differential expression of hundreds of genes across sham and nerve...
Citations
... The other upregulated gene in the OUM model, Serpina3n, encodes the serine protease inhibitor SerpinA3N, which is expressed in the brain and is associated with apoptosis and wound healing [50]. SerpinA3N has been identified as a pain-inhibiting factor in neuropathic pain models because it suppresses leukocyte elastase activity [51]. In this study, upregulation of these genes was observed in the TG in the OUM model. ...
Severe intraoral pain induces difficulty in eating and speaking, leading to a decline in the quality of life. However, the molecular mechanisms underlying intraoral pain remain unclear. Here, we investigated gene modulation in the trigeminal ganglion and intraoral pain-related behavior in a rat model of acetic acid-induced oral ulcerative mucositis. Oral ulceration was observed on day 2 after acetic acid treatment to the oral mucosa of male Wistar rats, causing spontaneous pain and mechanical allodynia. Deoxyribonucleic acid microarray analysis of trigeminal ganglion tissue indicated that Hamp (a hepcidin gene that regulates cellular iron transport) was the most upregulated gene. In the oral ulcerative mucositis model, the upregulation of Hamp was also induced in the ulcer region but not in the liver, with no increase in hepcidin levels in the plasma and saliva, indicating that hepcidin was produced locally in the ulcer region in the model. Systemic antibiotic pretreatment did not increase the mRNA levels of Hamp in the trigeminal ganglion and ulcer regions. Hepcidin injection into the oral mucosa enhanced neuronal excitability in response to noxious mechanical stimulation of the oral mucosa in trigeminal spinal subnucleus interpolaris/caudalis neurons. These results imply that oral ulcerative mucositis induces oral mucosal pain because of infectious inflammation of the ulcerative area and potentiates Hamp, which represents anti-bacterial and anti-peptidase gene expression in the ulcer region and trigeminal ganglion. The regulation of cellular iron transport by hepcidin is likely involved in oral ulcerative mucositis-induced pain.
... Polarization of resident macrophages in dorsal root ganglia and mitochondrial transfer from infiltrating macrophages to somata can confer sensitization of peripheral sensory neurons (11,12). Similarly, CD3+ T cells have also been described in the resolution pathway of inflammatory pain (13), and pharmacological blockade of T cell-derived leukocyte elastase significantly reduces the magnitude of behavioral hypersensitivity in a rodent model of neuropathic pain (14). Conversely, Na v 1.8+ sensory neurons play a key role in psoriasis and CD8+ T cell responses to viral infection (15). ...
... In the chronic constriction injury model of neuropathic pain, de novo neutrophil infiltration of ipsilateral dorsal root ganglia is observed with direct contact between polymorphonuclear granulocytes and neurons, despite no change in local expression of cytokine-induced neutrophil chemoattractant-1 (17). Moreover, nerve injury in mice leads to T cell and neutrophil infiltration of dorsal root ganglia and development of mechanical hypersensitivity that can be attenuated with inhibition of leukocyte-derived elastase (14,58). We observed de novo infiltration of neutrophils into lumbar DRG in primed mice that could be prevented with antibody depletion of circulating neutrophils (Fig. 3). ...
Fibromyalgia is a debilitating widespread chronic pain syndrome that occurs in 2 to 4% of the population. The prevailing view that fibromyalgia results from central nervous system dysfunction has recently been challenged with data showing changes in peripheral nervous system activity. Using a mouse model of chronic widespread pain through hyperalgesic priming of muscle, we show that neutrophils invade sensory ganglia and confer mechanical hypersensitivity on recipient mice, while adoptive transfer of immunoglobulin, serum, lymphocytes, or monocytes has no effect on pain behavior. Neutrophil depletion abolishes the establishment of chronic widespread pain in mice. Neutrophils from patients with fibromyalgia also confer pain on mice. A link between neutrophil-derived mediators and peripheral nerve sensitization is already established. Our observations suggest approaches for targeting fibromyalgia pain via mechanisms that cause altered neutrophil activity and interactions with sensory neurons.
... Both IL1rn and Saa3 are markers of sterile inflammation, are modulated by gut microbiota [76,77], and are associated with PN in both ob/ob and db/db mouse models [27,[78][79][80]. Mice with Ser-pin3 deficiency exhibit neuropathic pain, which can be reversed by exposure to exogenous Serpin3 [81]. We also identified Adam8, which stimulates axonal extension, as a novel target linking the gut microbiota to sciatic PN [82]. ...
Background
Peripheral neuropathy (PN) is a common complication in obesity, prediabetes, and type 2 diabetes, though its pathogenesis remains incompletely understood. In a murine high-fat diet (HFD) obesity model of PN, dietary reversal (HFD-R) to a low-fat standard diet (SD) restores nerve function and the nerve lipidome to normal. As the gut microbiome represents a potential link between dietary fat intake and nerve health, the current study assessed shifts in microbiome community structure by 16S rRNA profiling during the paradigm of dietary reversal (HFD-R) in various gut niches. Dietary fat content (HFD versus SD) was also correlated to gut flora and metabolic and PN phenotypes. Finally, PN-associated microbial taxa that correlated with the plasma and sciatic nerve lipidome and nerve transcriptome were used to identify lipid species and genes intimately related to PN phenotypes.
Results
Microbiome structure was altered in HFD relative to SD but rapidly reversed with HFD-R. Specific taxa variants correlating positively with metabolic health associated inversely with PN, while specific taxa negatively linked to metabolic health positively associated with PN. In HFD, PN-associated taxa variants, including Lactobacillus, Lachnoclostridium, and Anaerotruncus, also positively correlated with several lipid species, especially elevated plasma sphingomyelins and sciatic nerve triglycerides. Negative correlations were additionally present with other taxa variants. Moreover, relationships that emerged between specific PN-associated taxa variants and the sciatic nerve transcriptome were related to inflammation, lipid metabolism, and antioxidant defense pathways, which are all established in PN pathogenesis.
Conclusions
The current results indicate that microbiome structure is altered with HFD, and that certain taxa variants correlate with metabolic health and PN. Apparent links between PN-associated taxa and certain lipid species and nerve transcriptome-related pathways additionally provide insight into new targets for microbiota and the associated underlying mechanisms of action in PN. Thus, these findings strengthen the possibility of a gut-microbiome-peripheral nervous system signature in PN and support continuing studies focused on defining the connection between the gut microbiome and nerve health to inform mechanistic insight and therapeutic opportunities.
5A96nAvwbUx__NCbnk38-bVideo Abstract
... In addition, the presence of a small number of T lymphocytes in the dura, as well as memory CD4 and CD3 T cells in the subarachnoid space, has been reported (Coles et al., 2015;Schain et al., 2017). However, the relevance of the role of resident DCs and T cells in meningeal neurogenic inflammation in humans remains to be established, although their involvement has been widely shown in pre-clinical models of neuropathic and inflammatory pain (Kashem et al., 2015;Riol-Blanco et al., 2014;Vicuña et al., 2015). ...
The implications of neurogenic inflammation and neuroinflammation in the pathophysiology of migraine have been clearly demonstrated in preclinical migraine models involving several sites relevant in the trigemino-vascular system, including dural vessels and trigeminal endings, the trigeminal ganglion, the trigeminal nucleus caudalis as well as central trigeminal pain processing structures. In this context, a relevant role has been attributed over the years to some sensory and parasympathetic neuropeptides, in particular calcitonin gene neuropeptide, vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Several preclinical and clinical lines of evidence also support the implication of the potent vasodilator and messenger molecule nitric oxide in migraine pathophysiology. All these molecules are involved in vasodilation of the intracranial vasculature, as well as in the peripheral and central sensitization of the trigeminal system. At meningeal level, the engagement of some immune cells of innate immunity, including mast-cells and dendritic cells, and their mediators, has been observed in preclinical migraine models of neurogenic inflammation in response to sensory neuropeptides release due to trigemino-vascular system activation. In the context of neuroinflammatory events implicated in migraine pathogenesis, also activated glial cells in the peripheral and central structures processing trigeminal nociceptive signals seem to play a relevant role. Finally, cortical spreading depression, the pathophysiological substrate of migraine aura, has been reported to be associated with inflammatory mechanisms such as pro-inflammatory cytokine upregulation and intracellular signalling. Reactive astrocytosis consequent to cortical spreading depression is linked to an upregulation of these inflammatory markers. The present review summarizes current findings on the roles of immune cells and inflammatory responses in the pathophysiology of migraine and their possible exploitation in the view of innovative disease-modifying strategies.
... Importantly SerpinA3N is released in circulation in response to inflammation and is also known as an acute phase protein (Aslam and Yuan, 2020). Several studies have recently reported that SerpinA3N can regulate a wide range of biological processes (Haile et al., 2015, Tjondrokoesoemo et al., 2015, Vicuña et al., 2015, Gueugneau et al., 2018. In brief, SerpinA3N has been shown to induce neuroprotection in both in vitro and in vivo mouse models of neurodegenerative disease (Haile et al., 2015). ...
Aberrant hepatic lipid metabolism is the major cause of non-alcoholic fatty liver disease (NAFLD) and is associated with insulin resistance and type 2 diabetes. Serine (or cysteine) peptidase inhibitor, clade A, member 3N (SerpinA3N) is highly expressed in the liver, however its functional role in regulating NAFLD and associated metabolic disorders are not known. Male wildtype (WT) and hepatocyte Serpina3N knockout (HKO) mice were fed a control diet (CD), methionine and choline deficient (MCD) diet or high fat high sucrose (HFHS) diet to induce NAFLD and markers of lipid metabolism and glucose homeostasis were assessed. SerpinA3N protein was markedly induced in mice with fatty livers. Hepatic deletion of SerpinA3N attenuated steatosis which correlated with altered lipid metabolism genes, increased fatty acid oxidation activity and enhanced insulin signaling in mice with NAFLD. Additionally, SerpinA3N HKO mice had reduced epididymal white adipose tissue (eWAT) mass, leptin and insulin levels, improved glucose tolerance and enhanced insulin sensitivity which was associated with elevated insulin-like growth factor binding protein-1 (IGFBP1) and activation of the leptin receptor (LEPR)-STAT3 signaling pathway. Our findings provide a novel insight for the functional role for SerpinA3N in regulating NAFLD and glucose homeostasis.
... Here, activated astrocytes represent the main producers of SERPINA3 [101], whose expression is increased in response to IL-1 and TNF-α [102]. Previous experiments have shown that mice lacking serpina3n gene develop a more severe mechanical allodynia compared to wildtype mice [103]. Interestingly, Serpina3n was found to be upregulated in postmortem mid brain tissues from chronic cocaine abusers [104], and the astrogliosis induced by ischemic stroke-associated neuroinflammation was characterized by increased Serpina3n mRNA expression [105]. ...
... Interestingly, Serpina3n was found to be upregulated in postmortem mid brain tissues from chronic cocaine abusers [104], and the astrogliosis induced by ischemic stroke-associated neuroinflammation was characterized by increased Serpina3n mRNA expression [105]. Furthermore, Serpina3n is involved in neuropathic pain [103] and was actually found to be upregulated in dorsal root ganglia after peripheral nerve injury [106]. With this in mind, the down-regulation of Serpina3n expression in morphine-treated astrocytes following incubation with the medium of PEA-treated MCs can be considered a further proof of the ability of ultramicronized PEA to rebalance the astrocyte-MC crosstalk. ...
Persistent pain can be managed with opioids, but their use is limited by the onset of tolerance. Ultramicronized N-palmitoylethanolamine (PEA) in vivo delays morphine tolerance with mechanisms that are still unclear. Since glial cells are involved in opioid tolerance and mast cells (MCs) are pivotal targets of PEA, we hypothesized that a potential mechanism by which PEA delays opioid tolerance might depend on the control of the crosstalk between these cells. Morphine treatment (30 mM, 30 min) significantly increased MC degranulation of RBL-2H3 cells, which was prevented by pre-treatment with PEA (100 mM, 18 h), as evaluated by β-hexosaminidase assay and histamine quantification. The impact of RBL-2H3 secretome on glial cells was studied. Six-hour incubation of astrocytes with control RBL-2H3-conditioned medium, and even more so co-incubation with morphine, enhanced CCL2, IL-1β, IL-6, Serpina3n, EAAT2 and GFAP mRNA levels. The response was significantly prevented by the secretome from PEA pre-treated RBL-2H3, except for GFAP, which was further upregulated, suggesting a selective modulation of glial signaling. In conclusion, ultramicronized PEA down-modulated both morphine-induced MC degranulation and the expression of inflammatory and pain-related genes from astrocytes challenged with RBL-2H3 medium, suggesting that PEA may delay morphine tolerance, regulating MC-astrocyte crosstalk.
... Both hybridization-based microarray and RNA sequencing (RNA-seq) studies are commonly used to understand gene expression patterns in various physiological and pathological conditions. Accumulating studies have revealed that the dramatic transcriptional changes of many genes in spinal cord (SC) may drive neuropathic pain development and progression after nerve injury [10][11][12], implying that high-throughput transcriptome analyses have been successfully applied in identifying neuropathic pain-associated gene-expression signatures, which may be candidate markers for the diagnosis and treatment of neuropathic pain. In the current study, chronic constriction injury (CCI) rat model was established by loosely tying the sciatic nerve with 4 ligatures (4-0 chromic catgut) at the mid-thigh level. ...
Background
Growing evidence shows that C-Type Lectin Domain Containing 7A (Clec7a) may be involved into neuroinflammatory injury of various neurological diseases. However, its roles in neuropathic pain remain unclear.
Methods
A chronic constriction injury (CCI) rat model was constructed, and gene expression profilings in spinal cord tissues of CCI-insulted rats were detected by both microarray and RNA-seq studies. A series of bioinformatics analyses identified C/EBPβ-Clec7a to be a candidate axis involved into neuropathic pain. Then, its roles in mechanical allodynia, and pathological and molecular changes during CCI progression were determined by various gain-of-function and loss-of-function experiments in vivo and in vitro.
Results
Significant upregulation of Clec7a at both mRNA and protein levels were verified in spinal cord tissues of CCI-insulted rats. Clec7a knockdown markedly attenuated CCI-induced mechanical allodynia, obstructed Syk, ERK and JNK phosphorylation, inhibited NLRP3 inflammasome and caspase-1 activation, GSDMD cleavage, and consequently reduced the release of pro-inflammatory cytokines (all P < 0.05). Mechanically, the rat Clec7a promoter was predicted to bind with transcription factor C/EBPβ, confirmed by Luciferase assay and ChIP-qPCR. Both in vivo and in vitro assays demonstrated that C/EBPβ knockdown significantly suppressed CCI- or LPS/ATP-induced Clec7a upregulation, and subsequently reduced Syk, ERK and JNK phosphorylation, NLRP3 oligomerization, caspase-1 activation, GSDMD expression and pyroptosis, which were markedly reversed by the co-transfection of Clec7a expression vector.
Conclusions
This pre-clinical investigation reveals that C/EBPβ-Clec7a axis may be a potential target for relieving neuropathic pain through alleviating neuroinflammation, paving its way for clinical translation as a promising approach for neuropathic pain therapy.
... Here, we focus on some of the most recent studies related to NP. T cells in immune cells have a very important role to play, and studies have shown that mice lacking T cells completely lack the ability to produce neuropathic abnormal pain after a nerve injury (17). Furthermore, other studies have demonstrated that angiotensin 2 mediates the attenuation of neurological ectopic pain in the expression of invading macrophages at the site of nerve injury (18). ...
Neuropathic pain (NP) is a chronic health condition that presents a significant burden on patients, society, and even healthcare systems. However, in recent years, an emerging field in the treatment of neuropathic pain – optogenetic technology has dawned, heralding a new era in the field of medicine, and which has brought with it unlimited possibilities for studying the mechanism of NP and the treatment of research. Optogenetics is a new and growing field that uses the combination of light and molecular genetics for the first time ever. This rare combination is used to control the activity of living cells by expressing photosensitive proteins to visualize signaling events and manipulate cell activity. The treatments for NP are limited and have hardly achieved the desirable efficacy. NP differs from other types of pain, such as nociceptive pain, in that the treatments for NP are far more complex and highly challenging for clinical practice. This review presents the background of optogenetics, current applications in various fields, and the findings of optogenetics in NP. It also elaborates on the basic concepts of neuropathy, therapeutic applications, and the potential of optogenetics from the bench to the bedside in the near future.
... On the other hand, by secreting different kinds of mediators such as tumor necrosis factor alpha (TNF-α), nerve growth factor (NGF) and interleukin-1beta (IL-1β), some peripheral immune cells, such as macrophages, Schwann cells, lymphocytes and mast cells, enhance the excitability and sensibility of the primary sensory neurons and induce chronic pain, suggesting that interfering the interaction between macrophages and neurons may potentially help to alleviate chronic pain (Shu and Mendell 1999;Lim et al., 2015;Minnone et al., 2017;Aarão et al., 2018;Domoto et al., 2021). In addition, T cells, infiltrating into the dorsal root ganglion (DRG) of nerve injury model animals, were also reported to release leukocyte elastase (LE) and induce chronic pain, while this phenomenon could be inhibited by SerpinA3N inhibitor secreted by DRG neurons (Vicuña et al., 2015). Apart from peripheral immune cells, central glial cells including microglia and astrocytes were also proved to interact with neurons to regulate chronic pain, indicating that inhibiting the interaction between central glial cells and sensory neurons might also be a potential pathway for treating chronic pain (Ji et al., 2016). ...
Chronic pain is a disease of long-lasting pain with unpleasant feelings mediated by central and (or) peripheral sensitization, its duration usually lasts more than 3 months or longer than the expected recovery time. The patients with chronic pain are manifested with enhanced sensitivity to noxious and non-noxious stimuli. Due to an incomplete understanding of the mechanisms, patients are commonly insensitive to the treatment of first line analgesic medicine in clinic. Thus, the exploration of non-opioid-dependent analgesia are needed. Recent studies have shown that “sinomenine,” the main active ingredient in the natural plant “sinomenium acutum (Thunb.) Rehd. Et Wils,” has a powerful inhibitory effect on chronic pain, but its underlying mechanism still needs to be further elucidated. A growing number of studies have shown that various immune cells such as T cells, B cells, macrophages, astrocytes and microglia, accompanied with the relative inflammatory factors and neuropeptides, are involved in the pathogenesis of chronic pain. Notably, the interaction of the immune system and sensory neurons is essential for the development of central and (or) peripheral sensitization, as well as the progression and maintenance of chronic pain. Based on the effects of sinomenine on immune cells and their subsets, this review mainly focused on describing the potential analgesic effects of sinomenine, with rationality of regulating the neuroimmune interaction.
... Following sciatic nerve injury, the number of CD3-or CD4-positive T cells was increased in the spinal dorsal horn 9,10 . Besides, mechanical allodynia was not elicited in Rag1 -/mice and Rag2 -/mice which lack T cells and B cells 10,11 . In contrast, there are a few green fluorescence protein (GFP) and CD3 double-positive cells in the spinal cord after L4 spinal nerve transection in bone marrow chimeric mice that were transplanted GFP-positive bone marrow 12 . ...
... It has been reported that the adoptive transfer of T cells from mice with neuropathic pain elicited mechanical allodynia in naïve mice 9,27 , indicating the possible involvement of circulating activated T cells in neuropathic pain. Recent findings revealed the involvement of accumulated immune cells such as T cells and macrophages in the dorsal root ganglion (DRG) exacerbated neuropathic pain 11,28 . Considering the lack of blood-brain barrier in the trigeminal ganglion (TG) 29 , circulating T cells might be accumulated in the TG following skin re-incision in female rats and then potentiate neuronal activity in the TG. ...
Whisker pad skin incision in infancy causes the prolongation of mechanical allodynia after re-incision in adulthood. A recent study also proposed the importance of sex differences in pain signaling in the spinal cord. However, the sex difference in re-incision-induced mechanical allodynia in the orofacial region is not fully understood. In the rats that experienced neonatal injury in the whisker pad skin, the mechanical allodynia in the whisker pad was significantly prolonged after re-incision in adulthood compared to sham injury in infancy. No significant sex differences were observed in the duration of mechanical allodynia. The duration of mechanical allodynia in male rats was shortened by intracisternal administration of minocycline. However, minocycline had no effects on the duration of mechanical allodynia in female rats. In contrast, intracisternal administration of pioglitazone markedly suppressed mechanical allodynia in female rats after re-incision. Following re-incision, the number of peroxisome proliferator-activated receptor gamma (PPARgamma)-positive cells were reduced in the trigeminal spinal subnucleus caudalis (Vc) in female rats that experienced neonatal injury. Immunohistochemical analyses revealed that PPARgamma was predominantly expressed in Vc neurons. Pioglitazone increased the number of PPARgamma-positive Vc neurons in female rats whose whisker pad skin was incised in both infancy and adulthood stages. Pioglitazone also upregulated heme oxygenase 1 and downregulated NR1 subunit in the Vc in female rats after re-incision. Together, PPARgamma signaling in Vc neurons is a female-specific pathway for whisker pad skin incision-induced mechanical allodynia.
