Spared Nerve Injury Rats Research Articles

Analgesic effect and mechanism of electroacupuncture on SNI rats based on microglia-BDNF-neuron signal

To observe the effect of electroacupuncture (EA) at "Huantiao" (GB 30) and "Weizhong" (BL 40) on the activation of glial cells, the expression of brain-derived neurotrophic factor (BDNF), excitability and the number of dendritic spines of neurons in the spinal dorsal horn in rats with spared nerve injury (SNI) of sciatic nerve, and to explore the analgesic mechanism of EA on SNI. PartⅠ: Sixty SD rats were randomly divided into a sham operation group, a model group, an EA group and a sham EA group, 15 rats in each group. Except the sham operation group, the SNI rat model was established in the remaining groups. The rats in the sham operation group were only treated with incision without damaging the nerve. The rats in the EA group were treated with EA at "Huantiao" (GB 30) and "Weizhong" (BL 40) on the affected side, continuous wave, frequency of 2 Hz, current intensity of 1 mA, 30 minutes each time, once a day, for 14 days. The rats in the sham EA group were treated with EA at points 0.5 cm next to "Huantiao" (GB 30) and "Weizhong" (BL 40) on the affected side; the manipulation, EA parameters and treatment course were the same as the EA group. The latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex were detected 1 day before modeling and 3, 7 and 14 days after modeling. Fourteen days after modeling, Western blot was used to detect the protein expressions of ionized binding adapter junction protein 1 (Iba-1), glial fibrillary acidic protein (GFAP), BDNF and c-Fos in the spinal dorsal horn; the expressions of Iba-1 and c-Fos proteins in the spinal dorsal horn were detected by immunofluorescence staining; immunohistochemical method was used to detect the expression of GFAP protein in the spinal dorsal horn; Golgi staining was used to detect the number of dendritic spines in spinal dorsal horn neurons. PartⅡ: Thirty SD rats were randomly divided into a control group, a BDNF group and a BDNF+anti-TrkB group, 10 rats in each group. The control group was treated with intrathecal injection of 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and dimethyl sulfoxide (DMSO); the BDNF group was treated with intrathecal injection of 10 μg rat recombinant BDNF dissolved in 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and DMSO; the BDNF+anti-TrkB group was treated with intrathecal injection of 10 μg rat recombinant BDNF and 30 μg tyrosine kinase receptor B (TrkB) antibody dissolved in 10 μL mixture with 1︰1 of 0.9% sodium chloride solution and DMSO. The threshold of mechanical foot retraction reflex was detected 1 day before intrathecal injection and 1, 3 and 7 days after injection. Seven days after injection, the expression of c-Fos protein in the spinal dorsal horn was detected by Western blot and immunofluorescence staining. PartⅠ: Compared with the sham operation group, 3, 7 and 14 days after modeling, the latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex in the model group were decreased (P<0.05); 7 and 14 days after modeling, compared with the model group, the latency of thermal foot contraction reflex and the threshold of mechanical foot contraction reflex in the EA group were increased (P<0.05). The expressions of Iba-1, GFAP, BDNF, c-Fos proteins and the number of neuronal dendritic spines in the spinal dorsal horn in the model group were higher than those in the sham operation group (P<0.05); the expressions of Iba-1, BDNF, c-Fos proteins and the number of neuronal dendritic spines in the EA group were lower than those in the model group (P<0.05). PartⅡ: 3 and 7 days after intrathecal injection, the threshold of mechanical foot retraction reflex in the BDNF group was lower than that in the control group (P<0.05); the threshold of mechanical foot retraction reflex in the BDNF+anti-TrkB group was higher than that in the BDNF group (P<0.05). The expression of c-Fos protein in spinal dorsal horn in the BDNF group was higher than that in the control group (P<0.05); the expression of c-Fos protein in spinal dorsal horn in the BDNF+anti-TrkB group was lower than that in the BDNF group (P<0.05). The analgesic effect of EA at "Huantiao" (GB 30) and "Weizhong" (BL 40) on SNI rats may be related to inhibiting the activation of microglia in the dorsal horn of the spinal cord, thereby blocking the signal of microglia-BDNF-neuron, and finally reducing the excitability of neurons.

FGF 10 Inhibited Spinal Microglial Activation in Neuropathic Pain via PPAR-γ/NF-κB Signaling

Overactivated microglia in the spinal cord leads to neuropathic pain sensitivity. The FGF 10, a Fibroblast Growth Factor (FGFs) that is prevalent in neurons, has been demonstrated to suppress microglial polarization. The objective of this study was to investigate the role of FGF 10 in neuropathic pain and the underlying regulatory mechanisms. Immunofluorescence staining and western blot detection revealed that FGF 10 expression was upregulated in the ipsilateral spinal dorsal horn of Spared Nerve Injury (SNI) rat models and was mainly detected in neurons and microglia. To test the anti-microgliosis actions of FGF 10, SNI rats were intrathecally administered with different concentrations of recombinant FGF 10. Behavioral tests and immunostaining results showed that FGF 10 relieved hyperalgesia in SNI rats and inhibited microglial activity in the ipsilateral spinal dorsal horn in a dose-dependent manner. Besides, BV2 cells were cultured and treated with LPS to activate microglia to explore the underlying mechanisms of FGF 10-induced analgesic effects in vitro. As a result, FGF 10 administration suppressed the LPS-induced microglial augmentation in a dose-dependent manner, followed by increased PPAR-γ and decreased NFκB phosphorylation (p-NFκB) levels. Moreover, PPAR-γ agonist (pioglitazone) and antagonist (GW9662) were administrated into spinal cords of SNI rats, revealing that pioglitazone had similar anti-nociceptive and anti-microglial effects to FGF 10. Conversely, GW9662 reversed all beneficial effects of FGF 10 on SNI rats. In addition, phosphorylated levels of NFκB were reduced by pioglitazone or FGF 10 treatment but raised by GW9662 administration in FGF 10-treated SNI rats. Our findings show that FGF 10 has analgesic effects in rats after peripheral nerve injury and justify the role of PPAR-γ/NFκB signaling in FGF 10-regulated anti-microgliosis.

Positive interaction between GPER and β-alanine in the dorsal root ganglion uncovers potential mechanisms: mediating continuous neuronal sensitization and neuroinflammation responses in neuropathic pain

BackgroundThe pathogenesis of neuropathic pain and the reasons for the prolonged unhealing remain unknown. Increasing evidence suggests that sex oestrogen differences play a role in pain sensitivity, but few studies have focused on the oestrogen receptor which may be an important molecular component contributing to peripheral pain transduction. We aimed to investigate the impact of oestrogen receptors on the nociceptive neuronal response in the dorsal root ganglion (DRG) and spinal dorsal horn using a spared nerve injury (SNI) rat model of chronic pain.MethodsWe intrathecally (i.t.) administered a class of oestrogen receptor antagonists and agonists intrathecal (i.t.) administrated to male rats with SNI or normal rats to identify the main receptor. Moreover, we assessed genes identified through genomic metabolic analysis to determine the key metabolism point and elucidate potential mechanisms mediating continuous neuronal sensitization and neuroinflammatory responses in neuropathic pain. The excitability of DRG neurons was detected using the patch-clamp technique. Primary culture was used to extract microglia and DRG neurons, and siRNA transfection was used to silence receptor protein expression. Immunofluorescence, Western blotting, RT-PCR and behavioural testing were used to assess the expression, cellular distribution, and actions of the main receptor and its related signalling molecules.ResultsIncreasing the expression and function of G protein-coupled oestrogen receptor (GPER), but not oestrogen receptor-α (ERα) and oestrogen receptor-β (ERβ), in the DRG neuron and microglia, but not the dorsal spinal cord, contributed to SNI-induced neuronal sensitization. Inhibiting GPER expression in the DRG alleviated SNI-induced pain behaviours and neuroinflammation by simultaneously downregulating iNOS, IL-1β and IL-6 expression and restoring GABAα2 expression. Additionally, the positive interaction between GPER and β-alanine and subsequent β-alanine accumulation enhances pain sensation and promotes chronic pain development.ConclusionGPER activation in the DRG induces a positive association between β-alanine with iNOS, IL-1β and IL-6 expression and represses GABAα2 involved in post-SNI neuropathic pain development. Blocking GPER and eliminating β-alanine in the DRG neurons and microglia may prevent neuropathic pain development.

Effects of High-Voltage Pulsed Radiofrequency on the Ultrastructure and Nav1.7 Level of the Dorsal Root Ganglion in Rats With Spared Nerve Injury

ObjectivesTo investigate the analgesic effect of high-voltage pulsed radiofrequency (HV-PRF) on the dorsal root ganglion (DRG) for neuropathic pain induced by spared nerve injury (SNI) in rats, especially the influence of this treatment on the DRG ultrastructure and voltage-gated sodium channel 1.7 (Nav1.7) level in the DRG. Materials and MethodsOne hundred fifty adult male Sprague-Dawley rats were randomly divided into five groups: Sham, SNI, Free-PRF, standard-voltage PRF (SV-PRF), and HV-PRF. The 45V-PRF and 85V-PRF procedures applied to the left L5 DRG were performed in SV-PRF group and the HV-PRF group, respectively, on day 7 after SNI, whereas no PRF was concurrently delivered in Free-PRF group. The paw mechanical withdrawal threshold (PMWT) was detected before SNI (baseline) and on days 1, 3, 7, 8, 10, 14, and 21. The changes of left L5 DRG ultrastructure were analyzed with transmission electron microscopy on days 14 and 21. The expression levels of Nav1.7 in left L5 DRG were detected by immunofluorescence and Western blot. ResultsCompared with the Free-PRF group, PMWT in the SV-PRF group and HV-PRF group were both significantly increased after PRF (all p < 0.05). Meanwhile, the PMWT was significantly higher in the HV-PRF group than that in the SV-PRF group on days 14 and 21 (all p < 0.05). There were statistically significant differences between the SV-PRF and Free-PRF groups (p < 0.05). Similarly, statistically significant difference was found between the HV-PRF and Free-PRF groups (p < 0.05). Especially, comparison of the SV-PRF group and the HV-PRF group revealed statistically significant difference (p < 0.05). The Nav1.7 levels were significantly downregulated in the SV-PRF group and HV-PRF groups compared to that in the Free-PRF group (all p < 0.01). A significantly lower Nav1.7 level was also found in the HV-PRF group compared to that in the SV-PRF group (p < 0.05). ConclusionsThe HV-PRF produces a better analgesic effect than SV-PRF applied to the DRG in SNI rats. The underlying mechanisms may be associated with improving the histopathological prognosis and the downregulation of Nav1.7 levels in the DRG.

The analgesic effects of β-elemene in rats with neuropathic pain by inhibition of spinal astrocytic ERK activation.

Neuropathic pain takes a heavy toll on individual well-being, while current therapy is far from desirable. Herein, we assessed the analgesic effect of β-elemene, a chief component in the traditional Chinese medicine Curcuma wenyujin, and explored the underlying mechanisms at the level of spinal dorsal horn (SDH) under neuropathic pain. A spared nerve injury (SNI)-induced neuropathic pain model was established in rats. Intraperitoneal injection (i.p.) of β-elemene was administered for 21 consecutive days. Mechanical allodynia was explored by von Frey filaments. The activation of the mitogen-activated protein kinase (MAPK) family (including ERK, p38, and JNK) in spinal neurons, astrocytes, and microglia was evaluated using immunostaining 29 days after SNI surgery. The expression of GFAP, Iba-1, p-ERK, p-JNK, and p-p38 within the SDH was measured using immunoblotting. The levels of proinflammatory cytokines (including TNF-α, IL-1β, and IL-6) were measured with ELISA. The levels of oxidative stress indicators (including MDA, SOD, and GSH-PX) were detected using biochemical tests. Consecutive i.p. administration of β-elemene relieved SNI-induced mechanical allodynia (with an EC50 of 16.40 mg/kg). SNI significantly increased the expression of p-ERK in spinal astrocytes but not microglia on day 29. β-elemene reversed spinal astrocytic ERK activation and subsequent upregulation of proinflammatory cytokines in SNI rats, with no effect on the expression of p38 and JNK in spinal glia. β-elemene also exerted antioxidative effects by increasing the levels of SOD and GSH-PX and decreasing the level of MDA. Our results suggest that SNI induces robust astrocytic ERK activation within the SDH in the late phase of neuropathic pain. β-elemene exerts remarkable analgesic effects on neuropathic pain, possibly by inhibiting spinal astrocytic ERK activation and subsequent neuroinflammatory processes. Our findings suggest that β-elemene might be a promising analgesic for the treatment of chronic pain.

Lidocaine reduces pain behaviors by inhibiting the expression of Nav1.7 and Nav1.8 and diminishing sympathetic sprouting in SNI rats

Chronic neuropathic pain is a significant clinical challenge, and the mechanisms of neuropathic pain remain elusive. Previous studies have shown that spontaneous potential, which is triggered by Nav1.7 and Nav1.8 in the dorsal root ganglion (DRG), is crucial for the development of inflammatory and neuropathic pain. Functional coupling between the sympathetic nervous system and somatosensory nerves after a nerve injury has also been noted as an important factor in neuropathic pain. However, the relationship of sympathetic sprouting with Nav1.7 and Nav1.8 remains unclear. Therefore, we dynamically examined the mechanical withdrawal threshold (MWT), changes in Nav1.7 and Nav1.8, and sympathetic sprouting after lidocaine treatment in the spared nerve injury (SNI) model of rats. After lidocaine treatment, the MWT obviously increased, showing that hypersensitivity was significantly relieved and the abnormal expression of Nav1.7 and Nav1.8 caused by SNI was also significantly reduced. In addition, lidocaine distinctly inhibited sympathetic nerve sprouting and basket formation around the Nav1.7 and Nav1.8 neurons in the DRG. These results indicate that lidocaine may alleviate neuropathic pain by inhibiting the expression of Nav1.7 and Nav1.8, and diminishing sympathetic sprouting in DRG.

Intrathecal Administration of the α1 Adrenergic Antagonist Phentolamine Upregulates Spinal GLT-1 and Improves Mirror Image Pain in SNI Model Rats.

Mirror image pain (MIP) is a type of extraterritorial pain that results in contralateral pain or allodynia. Glutamate transporter-1 (GLT-1) is expressed in astrocytes and plays a role in maintaining low glutamate levels in the synaptic cleft. Previous studies have shown that GLT-1 dysfunction induces neuropathic pain. Our previous study revealed bilateral GLT-1 downregulation in the spinal cord of a spared nerve injury (SNI) rat. We hypothesized that spinal GLT-1 is involved in the mechanism of MIP. We also previously demonstrated noradrenergic GLT-1 regulation. Therefore, this study aimed to investigate the effect of an α1 adrenergic antagonist on the development of MIP. Rats were subjected to SNI. Changes in pain behavior and GLT-1 protein levels in the SNI rat spinal cords were then examined by intrathecal administration of the α1 adrenergic antagonist phentolamine, followed by von Frey test and western blotting. SNI resulted in the development of MIP and bilateral downregulation of GLT-1 protein in the rat spinal cord. Intrathecal phentolamine increased contralateral GLT-1 protein levels and partially ameliorated the 50% paw withdrawal threshold in the contralateral hind paw. Spinal GLT-1 upregulation by intrathecal phentolamine ameliorates MIP. GLT-1 plays a role in the development of MIPs.

P2X7 receptor-mediated release of microglial prostanoids and miRNAs correlates with reversal of neuropathic hypersensitivity in rats.

P2X7 receptor antagonists have potential for treating various central nervous system (CNS) diseases, including neuropathic pain, although none have been approved for clinical use. Reasons may include insufficient understanding of P2X7 receptor signalling in pain, and the lack of a corresponding preclinical mechanistic biomarker. Lu AF27139 is a highly selective and potent small molecule antagonist at rat, mouse and human forms of the P2X7 receptor, with excellent pharmacokinetic and CNS permeability properties. In the current experiments, we probed the utility of previously characterized and novel signalling cascades exposed to Lu AF27139 using cultured microglia combined with release assays. Subsequently, we assessed the biomarker potential of identified candidate molecules in the rat chronic constriction injury (CCI) model of neuropathic pain; study design limitations precluded their assessment in spared nerve injury (SNI) rats. Lu AF27139 blocked several pain-relevant pathways downstream of P2X7 receptors in vitro. At brain and spinal cord receptor occupancy levels capable of functionally blocking P2X7 receptors, it diminished neuropathic hypersensitivity in SNI rats, and less potently in CCI rats. Although tissue levels of numerous molecules previously linked to neuropathic pain and P2X7 receptor function (e.g. IL-6, IL-1β, cathepsin-S, 2-AG) were unaffected by CCI, Lu AF27139-mediated regulation of spinal PGE2 and miRNA (e.g. rno-miR-93-5p) levels increased by CCI aligned with its ability to diminish neuropathic hypersensitivity. We have identified a pain-relevant P2X7 receptor-regulated mechanism in neuropathic rats, which could hold promise as a translatable biomarker and by association enhance the clinical progression of P2X7 receptor antagonists in neuropathic pain. Sub-optimal translation of preclinical molecules has hindered the clinical development of novel mechanism of action analgesics. We have undertaken a comprehensive in vitro analysis of migroglial signalling mechanisms recruited upon P2X7 receptor activation, a number of which were shown to be modulated by a selective P2X7 receptor antagonist in a well characterized animal model of neuropathic pain. Subject to further confirmation in other neuropathic models, this opens up the possibility to investigate their clinical utility as potential pain biomarkers in patients.

Sirtuin 2 Alleviates Chronic Neuropathic Pain by Suppressing Ferroptosis in Rats.

Neuropathic pain (NP) is chronic and associated with poor effects of general analgesia. It affects patients’ health and quality of life. The apoptotic process of lipid peroxidation caused by iron overload is called ferroptosis, which may be associated with nervous system disease. A recent study has found that sirtuin 2 (SIRT2) achieves a neuroprotective effect by suppressing ferroptosis. Herein, we aimed to examine whether SIRT2 regulated spared nerve injury (SNI)-induced NP by suppressing ferroptosis in rats. A rat model of NP was induced in adult male Sprague-Dawley rats weighing 200–250 g. Mechanical allodynia was observed from the first day after SNI and continued for 14 days. Compared with age-matched control rats, the expression of SIRT2 and ferroportin 1 (FPN1) decreased in the L4-6 spinal cord of the SNI-induced NP rats. In addition, we observed that the levels of both iron and anti-acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) were significantly increased in the spinal cord after SNI, while the expression of glutathione peroxidase 4 (GPX4) was decreased. Furthermore, an intrathecal injection of SIRT2 overexpressed recombinant adenovirus, which upregulated the expression of SIRT2, attenuated mechanical allodynia, enhanced the level of FPN1, inhibited intracellular iron accumulation, and reduced oxidant stress levels, thereby reversing the changes to ACSL4 and GPX4 expression in the SNI rats. This evidence suggests that SIRT2-targeted therapeutics may help relieve the symptoms of chronic NP.

Reversal of neuropathic pain is associated with corticostriatal functional reorganization after nerve repair in the spared nerve injury model.

Following surgical repair after peripheral nerve injury, neuropathic pain diminishes in most patients but can persist in a small proportion of cases, the mechanism of which remains poorly understood. Based on the spared nerve injury (SNI), we developed a rat nerve repair (NR) model, where a delayed reconstruction of the SNI-injured nerves resulted in alleviating chronic pain-like behavior only in a subpopulation of rats. Multiple behavioral measures were assayed over 11-week presurgery and postsurgery periods (tactile allodynia, pain prick responses, sucrose preference, motor coordination, and cold allodynia) in SNI (n = 10), sham (n = 8), and NR (n = 12) rats. All rats also underwent resting-state functional magnetic resonance imaging under anesthesia at multiple time points postsurgery, and at 10 weeks, histology and retrograde labeling were used to calculate peripheral reinnervation. Behavioral measures indicated that at approximately 5 weeks postsurgery, the NR group separated to pain persisting (NR persisting, n = 5) and recovering (NR recovering, n = 7) groups. Counts of afferent nerves and dorsal root ganglion cells were not different between NR groups. Therefore, NR group differences could not be explained by peripheral reorganization. By contrast, large brain functional connectivity differences were observed between NR groups, where corticolimbic reorganization paralleled with pain recovery (repeated-measures analysis of variance, false discovery rate, P < 0.05), and functional connectivity between accumbens and medial frontal cortex was related both to tactile allodynia (nociception) and to sucrose preference (anhedonia) in the NR group. Our study highlights the importance of brain circuitry in the reversal of neuropathic pain as a natural pain-relieving mechanism. Further studies regarding the therapeutic potentials of these processes are warranted.

A Fully Implantable Miniaturized Liquid Crystal Polymer (LCP)-Based Spinal Cord Stimulator for Pain Control.

Spinal cord stimulation is a therapy to treat the severe neuropathic pain by suppressing the pain signal via electrical stimulation of the spinal cord. The conventional metal packaged and battery-operated implantable pulse generator (IPG) produces electrical pulses to stimulate the spinal cord. Despite its stable operation after implantation, the implantation site is limited due to its bulky size and heavy weight. Wireless communications including wireless power charging is also restricted, which is mainly attributed to the electromagnetic shielding of the metal package. To overcome these limitations, here, we developed a fully implantable miniaturized spinal cord stimulator based on a biocompatible liquid crystal polymer (LCP). The fabrication of electrode arrays in the LCP substrate and monolithically encapsulating the circuitries using LCP packaging reduces the weight (0.4 g) and the size (the width, length, and thickness are 25.3, 9.3, and 1.9 mm, respectively). An inductive link was utilized to wirelessly transfer the power and the data to implanted circuitries to generate the stimulus pulse. Prior to implantation of the device, operation of the pulse generator was evaluated, and characteristics of stimulation electrode such as an electrochemical impedance spectroscopy (EIS) were measured. The LCP-based spinal cord stimulator was implanted into the spared nerve injury rat model. The degree of pain suppression upon spinal cord stimulation was assessed via the Von Frey test where the mechanical stimulation threshold was evaluated by monitoring the paw withdrawal responses. With no spinal cord stimulation, the mechanical stimulation threshold was observed as 1.47 ± 0.623 g, whereas the stimulation threshold was increased to 12.7 ± 4.00 g after spinal cord stimulation, confirming the efficacy of pain suppression via electrical stimulation of the spinal cord. This LCP-based spinal cord stimulator opens new avenues for the development of a miniaturized but still effective spinal cord stimulator.

Intrathecal IGF2 siRNA injection provides long-lasting anti-allodynic effect in a spared nerve injury rat model of neuropathic pain.

Previous studies have shown an increase of insulin-like growth factor-2 (IGF2) in animal models of neuropathic pain. We aimed to examine the hypothesis that reducing the expression of IGF2 using intrathecal IGF2 small-interfering RNA (siRNA) would attenuate the development of neuropathic pain in rats after spared nerve injury (SNI). Male Wistar rats were divided into three groups: sham-operated group, in which surgery was performed to cut the muscles without injuring the nerves; SNI group, in which SNI surgery was performed to sever the nerves; and SNI + siRNA IGF2 group, in which SNI surgery was performed, and IGF2-siRNA was administered intrathecally 1 day after SNI. The rats were assessed for mechanical allodynia and cold allodynia 1 day before surgery (baseline), and at 2, 4, 6, 8, and 10 days after siRNA treatment. The rat spinal cord was collected for quantitative polymerase chain reaction and western blot analysis. Compared with the SNI group, rats that received IGF2 siRNA showed a significantly increased SNI-induced paw-withdrawal threshold to metal filament stimulation from Day 4 to Day 10 after SNI surgery. IGF2 siRNA significantly decreased the response duration from the acetone test from Day 2 to Day 10 following SNI surgery. SNI increased IGF2 mRNA expression on Day 2 and increased IGF2 protein expression on Day 8 and Day 10 in the spinal cord of the SNI rats. However, the above-mentioned effects of IGF2 mRNA and protein expression were significantly inhibited in the SNI + IGF2 siRNA group. We demonstrated that intrathecal administration of IGF2 siRNA provided significant inhibition of SNI-induced neuropathic pain via inhibition of IGF2 expression in the spinal cord. The analgesic effect lasted for 10 days. Further exploration of intrathecal IGF2 siRNA administration as a potential therapeutic strategy for neuropathic pain is warranted.

Pulsed Radiofrequency Upregulates Serotonin Transporters and Alleviates Neuropathic Pain-Induced Depression in a Spared Nerve Injury Rat Model.

Neuropathic pain (NP) is difficult to treat due to complex pathophysiological mechanisms. Pulsed radiofrequency (RRF) has been used widely with neuromodulation effect in refractory chronic pain treatment. A recent study found that PRF treatment may decrease chronic pain-related anxiety-depressant symptoms in patients, even though the mechanisms are unclear. Additionally, accumulated evidence has shown serotonin uptake is correlated with various neuropsychiatric diseases. Therefore, we investigated the effects and underlying mechanisms of PRF on depression-like behaviors, resulting from spared nerve injury (SNI)-induced NP. We examined the indexes of mechanical allodynia, cold allodynia, depression-like behavior, and blood cytokines by dynamic plantar aesthesiometry, acetone spray test, forced swimming test, and ProcartaPlex multiplex immunoassays in male Wistar rats, respectively. Serotonin transporters (SERTs) in rat brains were examined by using 4-[18F]-ADAM/PET imaging. We found that specific uptake ratios (SURs) of SERTs were significantly decreased in the brain regions of the thalamus and striatum in rats with SNI-induced NP and depression-like behaviors. Additionally, the decrease in SERT density was correlated with the development of a depression-like behavior indicated by the forced swimming test results and pronounced IL-6 cytokines. Moreover, we demonstrated that PRF application could modulate the descending serotoninergic pathway to relieve pain and depression behaviors.

The Occurrence of Pain-Induced Depression Is Different between Rat Models of Inflammatory and Neuropathic Pain.

Various pain conditions may be associated with depressed mood. However, the effect of inflammatory or neuropathic pain on depression-like behavior and its associated time frame has not been well established in rat models. This frontward study investigated the differences in pain behavior, depression-like behavior, and serotonin transporter (SERT) distribution in the brain between rats subjected to spared nerve injury (SNI)-induced neuropathic pain or complete Freund’s adjuvant (CFA)-induced inflammatory pain. A dynamic plantar aesthesiometer and an acetone spray test were used to evaluate mechanical and cold allodynia responses, and depression-like behavior was examined using a forced swimming test and sucrose preference test. We also investigated SERT expression by using positron emission tomography. We found that the inflammation-induced pain was less severe than neuropathic pain from days 3 to 28 after induced pain; however, the CFA-injected rats exhibited more noticeable depression-like behavior and had significantly reduced SERT expression in the brain regions (thalamus and striatum) at an early stage (on days 14, 21, and 28 in two groups of CFA-injected rats versus day 28 in SNI rats). We speculated that not only the pain response after initial injury but also the subsequent neuroinflammation may have been the crucial factors influencing depression-like behavior in rats.

Spared Nerve Injury Causes Sexually Dimorphic Mechanical Allodynia and Differential Gene Expression in Spinal Cords and Dorsal Root Ganglia in Rats

Neuropathic pain is more prevalent in women. However, females are under-represented in animal experiments, and the mechanisms of sex differences remain inadequately understood. We used the spared nerve injury (SNI) model in rats to characterize sex differences in pain behaviour, unbiased RNA-Seq and proteomics to study the mechanisms. Male and female rats were subjected to SNI- and sham-surgery. Mechanical and cold allodynia were assessed. Ipsilateral lumbar dorsal root ganglia (DRG) and spinal cord (SC) segments were collected for RNA-seq analysis with DESeq2 on Day 7. Cerebrospinal fluid (CSF) samples for proteomic analysis and DRGs and SCs for analysis of IB-4 and CGRP, and IBA1 and GFAP, respectively, were collected on Day 21. Females developed stronger mechanical allodynia. There were no differences between the sexes in CGRP and IB-4 in the DRG or glial cell markers in the SC. No CSF protein showed change following SNI. DRG and SC showed abundant changes in gene expression. Sexually dimorphic responses were found in genes related to T-cells (cd28, ctla4, cd274, cd4, prf1), other immunological responses (dpp4, c5a, cxcr2 and il1b), neuronal transmission (hrh3, thbs4, chrna4 and pdyn), plasticity (atf3, c1qc and reg3b), and others (bhlhe22, mcpt1l, trpv6). We observed significantly stronger mechanical allodynia in females and numerous sexually dimorphic changes in gene expression following SNI in rats. Several genes have previously been linked to NP, while some are novel. Our results suggest gene targets for further studies in the development of new, possibly sex-specific, therapies for NP.

Trifluoro-icaritin alleviates mechanical hypersensitivity and improves motor coordination and balance in rats with spared nerve injury-induced neuropathic pain

Neuropathic pain is still one of the unsolved public health problems worldwide. Although the current reagents can attenuate neuropathic pain to a certain extent, their clinical application is very limited owing to larger toxicity and serious side effects. Trifluoro-icaritin (ICTF) has been documented to possess profound anti-inflammatory and neuroprotective activities, but whether ICTF exerts an anti-nociceptive effect on neuropathic pain remains unknown. Here, a rat model of spared nerve injury (SNI)-induced neuropathic pain was used. SNI rats were administrated with ICTF (i.p.) once daily lasting for 21 days, and subsequently the pain-related behaviors were evaluated by applying mechanical or thermal pain threshold, CatWalk gait parameter, and rotarod test on day 1 before and day 1, 3, 7, 10, 14, and 21 after SNI surgery, respectively. The results showed that ICTF (0.5 mg/kg, 1.5 mg/kg, and 5.0 mg/kg, i.p.) treatment alleviated SNI-induced mechanical allodynia but not thermal hyperalgesia in a dose-dependent manner. After administration of ICTF at the most effective dose of 5.0 mg/kg to SNI rats, CatWalk gait analysis revealed that ICTF not only significantly enhanced gait parameters including max contact max intensity, max intensity, print area, and stand time but also decreased the swing time; Rotarod test further exhibited that ICTF could effectively prolong the time on rod and increase the rotating speed in SNI rats. Additionally, following ICTF (5.0 mg/kg) treatment of SNI rats for 21 consecutive days, the max contact max intensity was found to be positively correlated with the rotating speed. Taken together, ICTF successfully ameliorates mechanical hypersensitivity and improves the motor coordination and balance in SNI rats, suggesting that ICTF may be exploited as a potential candidate in the management of neuropathic pain.

Stereological Study of Changes of GABA-Immunoreactive Neurons in Spinal Dorsal Horn of SNI Rats.

Objective To observe the changes in the mechanical withdrawal threshold (MWT) and the proportion of GABA-immunoreactive neurons in spinal dorsal horn (SDH) of the spared nerve injury (SNI) rat model. Methods Thirty-six healthy male SD rats were randomly divided into a sham-operated group (group D, n = 18) and an SNI group (group S, n = 18). The left sciatic nerve trunk and three branches were exposed, two of which, known as tibial and the peroneal nerve, were ligated and cut off. The sural nerve was preserved to build the SNI model in group S. The left sciatic nerve trunk and three branches were only exposed in group D. MWT tests were performed on the medial and lateral sides of the rats' left hindpaw 1 day before surgery and at 7th, 14th, and 28th day after surgery. Results In group S, compared with the baseline measured 1 day before surgery, MWT on the medial and lateral sides of the rats' left hindpaw decreased significantly on the 7th, 14th, and 28th days after surgery (P < 0.05), while in group D, there was no statistically significant difference (P > 0.05). Compared with right SDH, there were not statistically significant reductions in the proportions of GABAergic neurons of left SDH on 7th and 28th day after SNI (P > 0.05); however, the proportion of GABAergic neurons in left SDH significantly decreased, compared with that in right side on 14th day after SNI (P < 0.05). On the same way, the proportions of GABAergic neurons on 7th, 14th, and 28th day after surgery were not statistically different (P > 0.05) in group D. Conclusion The SNI model could reduce the proportion of GABA-immunoreactive neurons in the rat's spinal dorsal horn on the nerve-injured side, and this change was lasting, which might be related to the transformation of the GABA-immunoreactive neurons.

Mammalian target of rapamycin signaling pathway is involved in synaptic plasticity of the spinal dorsal horn and neuropathic pain in rats by regulating autophagy.

Unveiling the etiology and the underlying mechanism of neuropathic pain, a poorly treated disease, is essential for the development of effective therapies. This study aimed to explore the role of mammalian target of rapamycin (mTOR) signaling in autophagy-mediated neuropathic pain. We established a spared nerve injury (SNI) model in adult male SD rats by ligating the common peroneal nerve and tibial, with the distal end cutoff. The paw withdrawal threshold (PWT) and C/A-fiber evoked field potentials were determined by electrophysiologic tests at day 0 (before operation), day 7 and day 14 postoperation, and SNI significantly increased field potentials (P < 0.05). Immunohistochemistry and western blots using spinal cord tissues showed that the expressions of GluR1, GluR2, Beclin-1, p62, mTOR and 4EBP1 were significantly increased after SNI (all P < 0.05), whereas the expressions of LC3 and LAMP2 were significantly decreased after SNI (all P < 0.05). Rapamycin efficiently counteracted the effect of SNI and restored the phenotypes to the level comparable to the sham control. In conclusion, rapamycin inhibits C/A-fiber-mediated long-term potentiation in the SNI rat model of neuropathic pain, which might be mediated by activation of autophagy signaling and downregulation of GluRs expression.

Long noncoding RNA LOC100911498 is a novel regulator of neuropathic pain in rats.

IntroductionNeuropathic pain (NP) is the most debilitating of all clinical pain syndromes and may be a consequence of dysfunction in the somatosensory nervous system. Unfortunately, the pathogenesis of NP is not fully understood yet and it cannot be cured totally. Long noncoding RNA (lncRNA) is a type of RNA molecule greater than 200 nucleotides, and dysregulated expression of lncRNAs play a critical role in the facilitation of NP. Previous study showed the expression level of LOC100911498 in the spinal cords of spared nerve injury (SNI) rats were increased. This research was aimed at exploring what role LOC100911498 plays in the pathophysiological process of NP.MethodsThe mechanical withdrawal threshold (MWT) of rats was measured by the von Frey test. The expression levels of P2X4 receptor (P2X4R), ionized calcium‐binding adaptor molecule 1 (Iba‐1), p‐p38 and brain‐derived neurotrophic factor (BDNF) in spinal cords were detected, respectively.ResultsOur results suggested that the level of LOC100911498 in SNI rats was markedly higher than that in the sham group; the MWT values in rats were treated with LOC100911498siRNA were increased, and the expression levels of P2X4R, Iba‐1, p‐p38 and BDNF in SNI+ LOC100911498siRNA group were reduced compared with those in the SNI group.ConclusionOur study indicated the effects lncRNA LOC100911498 siRNA exerted on NP were mediated by P2X4R on microglia in the spinal cords of rats. Further, LOC100911498 may be a novel positive regulator of NP by regulating the expression and function of the P2X4R.

Sirt2 in the Spinal Cord Regulates Chronic Neuropathic Pain Through Nrf2-Mediated Oxidative Stress Pathway in Rats

Reduction in Nrf2-mediated antioxidant response in the central nervous system plays an important role in the development and maintenance of neuropathic pain (NP). However, the mechanisms regulating Nrf2 activity in NP remain unclear. A recent in vitro study revealed that Sirt2, a member of the sirtuin family of proteins, affects antioxidant capacity by modulating Nrf2 activity. Here we examined whether central Sirt2 regulates NP through Nrf2-mediated oxidative stress pathway. In a rat model of spared nerve injury (SNI)-induced NP, mechanical allodynia and thermal hyperalgesia were observed on day 1 and up to day 14 post-SNI. The expression of Sirt2, Nrf2 and its target gene NQO1 in the spinal cord in SNI rats, compared with sham rats, was significantly decreased from day 7 and remained lower until the end of the experiment (day 14). The mechanical allodynia and thermal hyperalgesia in SNI rats were ameliorated by intrathecal injection of Nrf2 agonist tBHQ, which normalized expression of Nrf2 and NQO1 and reversed SNI-induced decrease in antioxidant enzyme superoxide dismutase (SOD) and increase in oxidative stress marker 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the spinal cord. Moreover, intrathecal injection of a recombinant adenovirus expressing Sirt2 (Ad-Sirt2) that upregulated expression of Sirt2, restored expression of Nrf2 and NQO1 and attenuated oxidative stress in the spinal cord, leading to improvement of thermal hyperalgesia and mechanical allodynia in SNI rats. These findings suggest that peripheral nerve injury downregulates Sirt2 expression in the spinal cord, which inhibits Nrf2 activity, leading to increased oxidative stress and the development of chronic NP.