Spinal Nerve Ligation Rats Research Articles

Contribution of AMPA Receptor-Mediated LTD in LA/BLA-CeA Pathway to Comorbid Aversive and Depressive Symptoms in Neuropathic Pain.

Comorbid anxiety and depressive symptoms in chronic pain are a common health problem, but the underlying mechanisms remain unclear. Previously, we have demonstrated that sensitization of the CeA neurons via decreased GABAergic inhibition contributes to anxiety-like behaviors in neuropathic pain rats. In this study, by using male Sprague Dawley rats, we reported that the CeA plays a key role in processing both sensory and negative emotional-affective components of neuropathic pain. Bilateral electrolytic lesions of CeA, but not lateral/basolateral nucleus of the amygdala (LA/BLA), abrogated both pain hypersensitivity and aversive and depressive symptoms of neuropathic rats induced by spinal nerve ligation (SNL). Moreover, SNL rats showed structural and functional neuroplasticity manifested as reduced dendritic spines on the CeA neurons and enhanced LTD at the LA/BLA-CeA synapse. Disruption of GluA2-containing AMPAR trafficking and endocytosis from synapses using synthetic peptides, either pep2-EVKI or Tat-GluA2(3Y), restored the enhanced LTD at the LA/BLA-CeA synapse, and alleviated the mechanical allodynia and comorbid aversive and depressive symptoms in neuropathic rats, indicating that the endocytosis of GluA2-containing AMPARs from synapses is probably involved in the LTD at the LA/BLA-CeA synapse and the comorbid aversive and depressive symptoms in neuropathic pain in SNL-operated rats. These data provide a novel mechanism for elucidating comorbid aversive and depressive symptoms in neuropathic pain and highlight that structural and functional neuroplasticity in the amygdala may be important as a promising therapeutic target for comorbid negative emotional-affective disorders in chronic pain.SIGNIFICANCE STATEMENT Several studies have demonstrated the high comorbidity of negative affective disorders in patients with chronic pain. Understanding the affective aspects related to chronic pain may facilitate the development of novel therapies for more effective management. Here, we unravel that the CeA plays a key role in processing both sensory and negative emotional-affective components of neuropathic pain, and LTD at the amygdaloid LA/BLA-CeA synapse mediated by GluA2-containing AMPAR endocytosis underlies the comorbid aversive and depressive symptoms in neuropathic pain. This study provides a novel mechanism for elucidating comorbid aversive and depressive symptoms in neuropathic pain and highlights that structural and functional neuroplasticity in the amygdala may be important as a promising therapeutic target for comorbid negative emotional-affective disorders in chronic pain.

Three-dimensional analysis of the characteristics of joint motion and gait pattern in a rodent model following spinal nerve ligation

BackgroundThe spinal nerve ligation (SNL) rat is well known as the most common rodent model of neuropathic pain without motor deficit. Researchers have performed analyses using only the von Frey and thermal withdrawal tests to evaluate pain intensity in the rat experimental model. However, these test are completely different from the neurological examinations performed clinically. We think that several behavioral reactions must be observed following SNL because the patients with neuropathic pain usually have impaired coordination of the motions of the right–left limbs and right–left joint motion differences. In this study, we attempted to clarify the pain behavioral reactions in SNL rat model as in patients. We used the Kinema-Tracer system for 3D kinematics gait analysis to identify new characteristic parameters of each joint movement and gait pattern.ResultsThe effect of SNL on mechanical allodynia was a 47 ± 6.1% decrease in the withdrawal threshold during 1–8 weeks post-operation. Sagittal trajectories of the hip, knee and ankle markers in SNL rats showed a large sagittal fluctuation of each joint while walking. Top minus bottom height of the left hip and knee that represents instability during walking was significantly larger in the SNL than sham rats. Both-foot contact time, which is one of the gait characteristics, was significantly longer in the SNL versus sham rats: 1.9 ± 0.15 s vs. 1.03 ± 0.15 s at 4 weeks post-operation (p = 0.003). We also examined the circular phase time to evaluate coordination of the right and left hind-limbs. The ratio of the right/left circular time was 1.0 ± 0.08 in the sham rats and 0.62 ± 0.15 in the SNL rats at 4 weeks post-operation.ConclusionsWe revealed new quantitative parameters in an SNL rat model that are directly relevant to the neurological symptoms in patients with neuropathic pain, in whom the von Frey and thermal withdrawal tests are not used at all clinically. This new 3D analysis system can contribute to the analysis of pain intensity of SNL rats in detail similar to human patients’ reactions following neuropathic pain.

IKBKB siRNA-Encapsulated Poly (Lactic-co-Glycolic Acid) Nanoparticles Diminish Neuropathic Pain by Inhibiting Microglial Activation.

Activation of nuclear factor-kappa B (NF-κB) in microglia plays a decisive role in the progress of neuropathic pain, and the inhibitor of kappa B (IκB) is a protein that blocks the activation of NF-κB and is degraded by the inhibitor of NF-κB kinase subunit beta (IKBKB). The role of IKBKB is to break down IκB, which blocks the activity of NF-kB. Therefore, it prevents the activity of NK-kB. This study investigated whether neuropathic pain can be reduced in spinal nerve ligation (SNL) rats by reducing the activity of microglia by delivering IKBKB small interfering RNA (siRNA)-encapsulated poly (lactic-co-glycolic acid) (PLGA) nanoparticles. PLGA nanoparticles, as a carrier for the delivery of IKBKB genes silencer, were used because they have shown potential to enhance microglial targeting. SNL rats were injected with IKBKB siRNA-encapsulated PLGA nanoparticles intrathecally for behavioral tests on pain response. IKBKB siRNA was delivered for suppressing the expression of IKBKB. In rats injected with IKBKB siRNA-encapsulated PLGA nanoparticles, allodynia caused by mechanical stimulation was reduced, and the secretion of pro-inflammatory mediators due to NF-κB was reduced. Delivering IKBKB siRNA through PLGA nanoparticles can effectively control the inflammatory response and is worth studying as a treatment for neuropathic pain.

Chronic Pain Produces Reversible Memory Deficits That Depend on Task Difficulty in Rats

Cognitive impairment associated with chronic pain remains relatively poorly understood. Use of analgesic drugs and often present co-morbidities in patients can preclude conclusions of causative relationships between chronic pain and cognitive deficits. Here, the impact of pain resulting from spinal nerve ligation (SNL) injury in rats on short and long-term memory was assessed in the novel object recognition task. To understand if chronic pain seizes the limited cognitive resources that are available at any given time, task difficulty was varied by using either very different (ie, easy task) or similar (ie, difficult task) pairs of objects. Nerve-injured, male rats exhibited no short or long-term memory deficits under easy task conditions. However, unlike sham-operated controls, injured rats showed deficits in both short and long-term memory by failing to differentiate similar objects in the difficult task version. In SNL rats, duloxetine produced anti-allodynic effects and ameliorated long-term memory deficits in the difficult task suggesting benefits of pain relief possibly complemented by noradrenergic mediated cognitive enhancement. Together these data suggest chronic pain reversibly takes up a significant amount of limited cognitive resources, leaving sufficient available for easy, but not difficult, tasks. PerspectiveMemory deficits in a rat model of chronic pain were only seen when the cognitive load was high, ie, in a difficult task. Acute treatment with duloxetine was sufficient to relieve memory deficits, suggesting chronic pain induces memory deficits by seizing limited cognitive resources to the detriment of task-related stimuli.

Intracerebroventricular injection of propranolol blocked analgesic and neuroprotective effects of resveratrol following L5 spinal nerve ligation in rat.

Resveratrol as a natural polyphenolic agent can alleviate neuropathic pain symptoms. The mechanism of analgesic activity of resveratrol is far from clear. The current study examine whether analgesic activity of resveratrol is mediated by its neuroprotective and anti-oxidant activity in the neuropathic pain. We further examine whether analgesic activity of resveratrol is mediated by β-adrenoceptors in the brain. Neuropathic pain induced by L5 spinal nerve ligation (SNL). Male Wistar rats assigned into sham, SNL, SNL+resveratrol (40μg/5μL), and SNL+resveratrol+propranolol (a non-selective β-adrenoceptor antagonist, 30μg/5μL) groups. Drugs injected intracerebroventricular (ICV) at day SNL surgery and daily for 6 days following SNL. Thermal allodynia and anxiety examined on days of -1, 2, 4, and 6 following SNL. Electrophysiological study performed on day 6 following SNL for evaluation of resveratrol effects on sciatic nerve conduction velocity (NCV). The activity of catalase (Cat) and superoxide dismutase (SOD) enzymes in the brain assessed on days 6 following SNL. Resveratrol significantly decreased thermal allodynia (and not anxiety) in all experimental days. Additionally, resveratrol significantly increased NCV, and also normalized the disrupted Cat and SOD activities following neuropathic pain. Furthermore, propranolol significantly blocked the analgesic and neuroprotective effects of resveratrol. It is suggested that the analgesic effects of resveratrol is mediated by its neuroprotective and antioxidant activities in the neuropathic rats. Furthermore, propranolol blocked the analgesic and neuroprotective effects of resveratrol.

Protopanaxadiol alleviates neuropathic pain by spinal microglial dynorphin A expression following glucocorticoid receptor activation.

New remedies are required for the treatment of neuropathic pain due to insufficient efficacy of available therapies. This study provides a novel approach to develop painkillers for chronic pain treatment. The rat formalin pain test and spinal nerve ligation model of neuropathic pain were used to evaluate antinociception of protopanaxadiol. Primary cell cultures, immunofluorescence staining, and gene and protein expression were also performed for mechanism studies. Gavage protopanaxadiol remarkably produces pain antihypersensitive effects in neuropathic pain, bone cancer pain and inflammatory pain, with efficacy comparable with gabapentin. Long-term PPD administration does not induce antihypersensitive tolerance, but prevents and reverses the development and expression of morphine analgesic tolerance. Oral protopanaxadiol specifically stimulates spinal expression of dynorphin A in microglia but not in astrocytes or neurons. Protopanaxadiol gavage-related pain antihypersensitivity is abolished by the intrathecal pretreatment with the microglial metabolic inhibitor minocycline, dynorphin antiserum or specific κ-opioid receptor antagonist GNTI. Intrathecal pretreatment with glucocorticoid receptor)antagonists RU486 and dexamethasone-21-mesylate, but not GPR-30 antagonist G15 or mineralocorticoid receptor antagonist eplerenone, completely attenuates protopanaxadiol-induced spinal dynorphin A expression and pain antihypersensitivity in neuropathic pain. Treatment with protopanaxadiol, the glucocorticoid receptor agonist dexamethasone and membrane-impermeable glucocorticoid receptor agonist dexamethasone-BSA in cultured microglia induces remarkable dynorphin A expression, which is totally blocked by pretreatment with dexamthasone-21-mesylate. All the results, for the first time, indicate that protopanaxadiol produces pain antihypersensitivity in neuropathic pain probably through spinal microglial dynorphin A expression after glucocorticoid receptor activation and hypothesize that microglial membrane glucocorticoid receptor/dynorphin A pathway is a potential target to discover and develop novel painkillers in chronic pain.

Dorsal root ganglion stimulation of injured sensory neurons in rats rapidly eliminates their spontaneous activity and relieves spontaneous pain.

Dorsal root ganglion field stimulation (GFS) relieves evoked and spontaneous neuropathic pain by use-dependent blockade of impulse trains through the sensory neuron T-junction, which becomes complete within less than 1 minute for C-type units, also with partial blockade of Aδ units. We used this tool in the spinal nerve ligation (SNL) rat model to selectively block sensory neuron spontaneous activity (SA) of axotomized neurons at the fifth lumbar (L5) level vs blockade of units at the L4 level that remain uninjured but exposed to inflammation. In vivo dorsal root single-unit recordings after SNL showed increased SA in L5 units but not L4 units. Ganglion field stimulation blocked this SA. Ganglion field stimulation delivered at the L5 dorsal root ganglion blocked mechanical hyperalgesia behavior, mechanical allodynia, and ongoing spontaneous pain indicated by conditioned place preference, whereas GFS at L4 blocked evoked pain behavior but not spontaneous pain. In vivo single-unit recordings of spinal cord dorsal horn (DH) wide-dynamic-range neurons showed elevated SA after SNL, which was reduced by GFS at the L5 level but not by GFS at the L4 level. In addition, L5 GFS, but not L4 GFS, increased mechanical threshold of DH units during cutaneous mechanical stimulation, while L5 GFS exceeded L4 GFS in reducing evoked firing rates. Our results indicate that SA in injured neurons supports increased firing of DH wide-dynamic-range neurons, contributing to hyperalgesia, allodynia, and ongoing pain. Ganglion field stimulation analgesic effects after nerve injury are at least partly attributable to blocking propagation of this SA.

Effect of electroacupuncture on angiopoietin-1 in spinal cord of rats with neuropathic pain

To observe the effect of electroacupuncture (EA) on the behavior, histomorphology and the expression of angiopoietin-1 (Angpt-1) in rats with spinal nerve injury, so as to explore its mechanism on neuropathic pain. Forty-five male SD rats were randomly divided into sham, model and EA groups (n=15 rats in each group). Spinal nerve ligation (SNL) of the L5 lumbar vertebra was performed to establish a rat model of neuropathic pain. The rats in the EA group were given EA at "Zusanli" (ST36) and "Kunlun" (BL60) of the operation side with continuous wave at a frequency of 2 Hz and an intensity of 1.5 mA once a day, 30 minutes each time for 7 days. The sham group only exposed L5 spinal nerves without ligation. Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were observed and recorded before modeling and on days 3,5,7,10,12 and 14 after modeling. L4-L6 segments of spinal cord were taken and the morphological changes of spinal dorsal horn were observed by HE staining. The changes of spinal dorsal horn nerve fiber structure were observed by silver plating staining. Angpt-1 expression was detected by Western blot and immunohistochemistry. Compared with the sham group, the model group had significant reductions in MWT and TWL at each time point (P<0.01); compared with the model group, the EA group had significant increases in MWT and TWL on days 10,12 and 14 after intervention (P<0.05, P<0.01). HE staining showed that in the model group, the spinal dorsal horn showed degeneration and necrosis of neurons, nuclear fixation and shrinkage, and loose surrounding tissues. The degree of tissue damage of the EA group was milder than that of the model group. The silver staining results showed the model group had obvious neuronal fibrillary tangles, while there were fewer neuronal fibrillary tangles in the EA group. Compared with the sham group, the Angpt-1 expression in the model group was significantly decreased (P<0.01), and compared with the model group, the EA group had a significant increase in the expression of Angpt-1 (P<0.01). EA can promote the recovery of nerve function in SNL rats by up-regulating Angpt-1 expression.

S100A4 in Spinal Substantia Gelatinosa from Dorsal Root Ganglia Modulates Neuropathic Pain in a Rodent Spinal Nerve Injury Model.

PurposeTo detect the spatio-temporal expression of S100A4 in a spinal nerve ligation (SNL) rat model. Also to figure out which other molecules directly interact with S100A4 to explore the possible mechanisms which might be involved in neuropathic pain.MethodsSeven-week-old male SD rats were used for the SNL model construction. Immunofluorescence and Western blotting were used to detect the spatio-temporal expression of S100A4 in the model. S100A4 was co-labeled with a number of related molecules and marker molecules that can distinguish between cell types. After intrathecal injection of S100A4 neutralizing antibody, the behavioral changes of SNL rats were recorded, and molecular changes compared. The direct interaction between S100A4 and other related molecules was verified by co-immunoprecipitation (co-IP) to explore its possible mechanism.ResultsAfter spinal nerve ligation, the content of S100A4 in the dorsal root ganglion (DRG) and spinal dorsal horn increased significantly. Intrathecal injection of S100A4 neutralizing antibody could effectively relieve the mechanical pain in rats. co-IP revealed a direct interaction between S100A4 and RAGE.ConclusionThe content of S100A4 in the DRG and spinal dorsal horn of SNL rats increased, compared with that of the control group. Intrathecal injection of S100A4 neutralizing antibody could effectively relieve the mechanical pain in SNL rats. S100A4 may be involved in the production of neuropathic pain through RAGE or other ways, but the specific mechanism needs to be further studied.

Sinomenine alleviates dorsal root ganglia inflammation to inhibit neuropathic pain via the p38 MAPK/CREB signalling pathway

The objective of study was to investigate the inhibitory effect of sinomenine on neuropathic pain on dorsal root ganglia (DRG). The DRG cell line and spinal nerve ligation (SNL) model were used in this study. The effect of sinomenine on the cell viability was examined by MTT assay. The expression of p38 MAPK, NF-κB, c-fos, SP and TNF-α was detected by using immunofluorescence and immunohistochemistry assay. We also assessed the level of p-CaMKII, COX-2, p-CREB, IL-17A, TLR4 and IL-1β via western blotting and RT-qPCR. Compared to the controls, sinomenine showed a protective effect on TNF-α-induced apoptosis on DRG cells in a dose-dependent manner, with an increase of cell viability and a decrease of reactive oxygen species level as well as LDH release. Parallelly, sinomenine treatment significantly reduced the expression of various factors related to stress and inflammation, including p38 MAPK, NF-κB, c-fos, p-CAMKII, COX-2, p-CREB, TLR4 and IL-17A in DRG cells in vitro. Furthermore, we found that administration of sinomenine significantly reduced mechanical withdrawal threshold and thermal withdrawal latency and inhibited the inflammation and activation of p38 signaling in SNL rats. It is noting that combined therapy of sinomenine and pulsed radiofrequency exhibited higher efficacy of dorsal root ganglia inflammation than single treatment as well as the combination of oxycodone and pulsed radiofrequency. Sinomenine inhibited the apoptosis of DRG cell by regulating p38 MAPK/CREB signalling pathway, which provides evidence to alleviate neuropathic pain in clinic.

Electroacupuncture improves neuronal plasticity through the A2AR/cAMP/PKA signaling pathway in SNL rats

Improvements in neuronal plasticity are considered to be conducive to recovery from neuropathic pain. Electroacupuncture (EA) is regarded as an effective rehabilitation method for neuropathic pain. However, the effects and potential mechanism associated with EA-induced repair of hyperesthesia are not fully understood. Evidence has suggested that the adenosine A2A receptor (A2AR) and the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway play an important role in improving neuropathic pain. Here, we examined the function of EA in promoting neuronal plasticity in spinal nerve ligation (SNL) rats. The A2AR antagonist SCH58261, A2AR agonist 2-p-(2-carboxyethyl)phenethylamino-50-N-ethylcarboxamido adenosine HCl (CGS21680) and A2AR siRNA were used to confirm the relationship between A2AR and the cAMP/PKA pathway as well as the effects of A2AR on EA-induced improvements in neurobehavioral state and neuronal plasticity. Mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), HE staining, Western blotting, RT-PCR, immunofluorescence, enzyme-linked immunosorbent assay, Nissl staining, silver staining, Golgi-Cox staining and transmission electron microscopy were used to evaluate the changes in neurobehavioral performance, protein expression, neuronal structure and dendrites/synapses. The results showed that EA and CGS21680 improved the behavioral performance, neuronal structure and dendritic/synaptic morphology of SNL rats, consistent with higher expression levels of A2AR, cAMP and PKA. In contrast to the positive effects of EA, SCH58261 inhibited dendritic growth and promoted dendritic spine/synaptic remodeling. In addition, the EA-induced improvement in neuronal plasticity was inhibited by SCH58261 and A2AR siRNA, consistent with lower expression levels of A2AR, cAMP and PKA, and worse behavioral performance. These results indicate that EA suppresses SNL-induced neuropathic pain by improving neuronal plasticity via upregulating the A2AR/cAMP/PKA signaling pathway.

Electro-acupuncture Suppresses AXL Expression in Dorsal Root Ganglion Neurons and Enhances Analgesic Effect of AXL Inhibitor in Spinal Nerve Ligation Induced-Neuropathic Pain Rats.

Electro-acupuncture (EA) has been used for clinic analgesia for many years. However, its mechanisms are not fully understood. We recently reported that AXL, a tyrosine kinase receptor, contributes to the peripheral mechanism of neuropathic pain. We here aim to figure out the significance of EA on neuropathic pain mediated by AXL in dorsal root ganglion (DRG). Spinal nerve ligation (SNL) was used as a neuropathic pain model. EA was applied at ''Huantiao'' (GB-30) and ''Yanglingquan'' (GB-34) acupoints for 30min daily from day 7 to day 10 after SNL. EA not only gradually attenuated SNL-induced mechanical allodynia, but also suppressed the expression of phosphorylated AXL (p-AXL) and AXL in injured DRGs of SNL rats examined by western blotting and immunofluorescence. Moreover, intrathecal injection of the subthreshold dose of AXL inhibitor TP0903, significantly prolonged the analgesic time of single EA treatment and enhanced the analgesic effect of repeated EA treatments, suggesting a synergic effect of EA and AXL inhibitor. These results indicate that AXL signaling underlies EA analgesia and combination of AXL inhibitor and EA might be a new strategy for clinic analgesia on neuropathic pain.

Electroacupuncture may alleviate neuropathic pain via suppressing P2X7R expression.

Neuropathic pain is a severe problem that is difficult to treat clinically. Reducing abnormal remodeling of dendritic spines/synapses and increasing the anti-inflammatory effects in the spinal cord dorsal horn are potential methods to treat this disease. Previous studies have reported that electroacupuncture (EA) could increase the pain threshold after peripheral nerve injury. However, the underlying mechanism is unclear. P2X7 receptors (P2X7R) mediate the activation of microglia and participate in the occurrence and development of neuropathic pain. We hypothesized that the effects of EA on relieving pain may be related to the downregulation of the P2X7R. Spinal nerve ligation (SNL) rats were used as a model in this experiment, and 2'(3')-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a P2X7R agonist. We found that EA treatment decreased dendritic spine density, inhibited synaptic reconstruction and reduced inflammatory response, which is consistent with the decrease in P2X7R expression as well as the improved neurobehavioral performance. In contrast to the beneficial effects of EA, BzATP enhanced abnormal remodeling of dendritic spines/synapses and inflammation. Furthermore, the EA-mediated positive effects were reversed by BzATP, which is consistent with the increased P2X7R expression. These findings indicated that EA improves neuropathic pain by reducing abnormal dendritic spine/synaptic reconstruction and inflammation via suppressing P2X7R expression.

Electroacupuncture effects on the P2X4R pathway in microglia regulating the excitability of neurons in the substantia gelatinosa region of rats with spinal nerve ligation

Electroacupuncture (EA) has been used to treat neuropathic pain induced by peripheral nerve injury (PNI) by applying an electrical current to acupoints with acupuncture needles. However, the mechanisms by which EA treats pain remain indistinct. High P2X4 receptor (P2X4R) expression levels demonstrate a notable increase in hyperactive microglia in the ipsilateral spinal dorsal horn following PNI. In order to demonstrate the possibility that EA analgesia is mediated in part by P2X4R in hyperactive microglia, the present study performed mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) tests in male Sprague-Dawley rats that had undergone spinal nerve ligation (SNL). The expression levels of spinal P2X4R were determined using reverse transcription-quantitative PCR, western blotting analysis and immunofluorescence staining. Furthermore, spontaneous excitatory postsynaptic currents (sEPSCs) were recorded using whole-cell patch clamp to demonstrate the effect of EA on synaptic transmission in rat spinal substantia gelatinosa (SG) neurons. The results of the present study demonstrated that EA increased the MWT and TWL and decreased overexpression of P2X4R in hyperactive microglia in SNL rats. Moreover, EA attenuated the frequency of sEPSCs in SG neurons in SNL rats. The results of the present study indicate that EA may mediate P2X4R in hyperactive spinal microglia to inhibit nociceptive transmission of SG neurons, thus relieving pain in SNL rats.

Effect of Massage on the TLR4 Signalling Pathway in Rats with Neuropathic Pain.

This study set out to investigate the effect of massage on the Toll-like receptor 4 (TLR4) signalling pathway in the dorsal root ganglia of rats that had undergone spinal nerve ligation (SNL), with the hypothesis that massage could be used as an analgesic. Forty female SD rats were randomly divided into 5 groups: the control group, sham-operated group, model group, sham massage group, and massage group. There were 8 rats in each group. SNL rat models were established in the model group, sham massage group, and massage group. Rats in the sham-operated group underwent surgery to expose the vertebral nerves, but no further procedures were performed. The control group consisted of intact animals. The rats in the massage group underwent massage using a massage simulation machine once a day for 14 d in succession; the hind limbs of the rats in the sham massage group were gently touched with a cloth bag once a day for 14 continuous days. The rats in the control group, the sham-operated group, and the model group did not receive any intervention and were observed for 14 d. Paw withdrawal thermal latency (PWTL) and paw withdrawal mechanical threshold (PWMT) of rats in each group were detected 1 d before modelling and at 1, 3, 7, and 14 d after modelling. Fourteen days after modelling, the expression levels of TLR4, IRAK1, TRAF6, TNF-α, and IL-6 were detected in all rats. The PWTL and PWMT of SNL rats were decreased, while these parameters were elevated after massage. SNL rats showed higher levels of TLR4, IRAK1, TRAF6, IL-6, and TNF-α, and massage effectively lowered the expression levels of these molecules. Inhibiting activation of the TLR4 signalling pathway, which can reduce the release of inflammatory factors, may be one mechanism by which massage treats neuropathic pain.

Cytokine activin C ameliorates chronic neuropathic pain in peripheral nerve injury rodents by modulating the TRPV1 channel.

The cytokine activin C is mainly expressed in small-diameter dorsal root ganglion (DRG) neurons and suppresses inflammatory pain. However, the effects of activin C in neuropathic pain remain elusive. Male rats and wild-type and TRPV1 knockout mice with peripheral nerve injury - sciatic nerve axotomy and spinal nerve ligation in rats; chronic constriction injury (CCI) in mice - provided models of chronic neuropathic pain. Ipsilateral lumbar (L)4-5 DRGs were assayed for activin C expression. Chronic neuropathic pain animals were treated with intrathecal or locally pre-administered activin C or the vehicle. Nociceptive behaviours and pain-related markers in L4-5 DRGs and spinal cord were evaluated. TRPV1 channel modulation by activin C was measured. Following peripheral nerve injury, expression of activin βC subunit mRNA and activin C protein was markedly up-regulated in L4-5 DRGs of animals with axotomy, SNL or CCI. [Correction added on 26 November 2020, after first online publication: The preceding sentence has been corrected in this current version.] Intrathecal activin C dose-dependently inhibited neuropathic pain in spinal nerve ligated rats. Local pre-administration of activin C decreased neuropathic pain, macrophage infiltration into ipsilateral L4-5 DRGs and microglial reaction in L4-5 spinal cords of mice with CCI. In rat DRG neurons, activin C enhanced capsaicin-induced TRPV1 currents. Pre-treatment with activin C reduced capsaicin-evoked acute hyperalgesia and normalized capsaicin-evoked persistent hypothermia in mice. Finally, the analgesic effect of activin C was abolished in TRPV1 knockout mice with CCI. Activin C inhibits neuropathic pain by modulating TRPV1 channels, revealing potential analgesic applications in chronic neuropathic pain therapy.

Ubiquitin-mediated receptor degradation contributes to development of tolerance to MrgC agonist-induced pain inhibition in neuropathic rats.

Agonists to subtype C of the Mas-related G-protein-coupled receptors (MrgC) induce pain inhibition after intrathecal (i.t.) administration in rodent models of nerve injury. Here, we investigated whether tolerance develops after repeated MrgC agonist treatments and examined the underlying mechanisms. In animal behavior studies conducted in male rats at 4 to 5 weeks after an L5 spinal nerve ligation (SNL), the ability of dipeptide MrgC agonist JHU58 (0.1 mM, 10 μL, i.t.) to inhibit mechanical and heat hypersensitivity decreased after 3 days of treatment with a tolerance-inducing dose (0.5 mM, 10 μL, i.t., twice/day). In HEK293T cells, acute treatment with JHU58 or BAM8-22 (a large peptide MrgC agonist) led to MrgC endocytosis from the cell membrane and later sorting to the membrane for reinsertion. However, chronic exposure to JHU58 increased the coupling of MrgC to β-arrestin-2 and led to the ubiquitination and degradation of MrgC. Importantly, pretreatment with TAK-243 (0.2 mM, 5 μL, i.t.), a small-molecule inhibitor of the ubiquitin-activating enzyme, during tolerance induction attenuated the development of tolerance to JHU58-induced inhibition of mechanical and heat hypersensitivity in SNL rats. Interestingly, morphine analgesia was also decreased in SNL rats that had become tolerant to JHU58, suggesting a cross-tolerance. Furthermore, i.t. pretreatment with TAK-243, which reduced JHU58 tolerance, also attenuated the cross-tolerance to morphine analgesia. These findings suggest that tolerance can develop to MrgC agonist-induced pain inhibition after repeated i.t. administrations. This tolerance development to JHU58 may involve increased coupling of MrgC to β-arrestin-2 and ubiquitin-mediated receptor degradation.

Adipose Tissue-Derived Stem Cells Alleviate Cold Allodynia in a Rat Spinal Nerve Ligation Model of Neuropathic Pain.

Neuropathic pain caused by lesions or nervous system dysfunction is a neuroimmune disease with limited therapeutic options. Adipose tissue-derived stem cells (ASCs) are multipotent mesenchymal stem cells with potent immunosuppressive properties, and their use as novel cell-based therapeutics have been proposed in many immune diseases. However, the analgesic effect and efficacy of ASCs to treat neuropathic pain remain unclear. This study, thus, investigated whether ASCs or ASC-derived culture medium can relieve neuropathic pain behaviors (i.e., mechanical and cold allodynia) in a rat model with L5 spinal nerve ligation. Intrathecal injection of ASCs significantly reduced cold allodynia, but not mechanical allodynia. Importantly, cold allodynia was completely reversed in rats with repeated injections of ASCs. In contrast, intrathecal injection of ASC-derived culture medium or retro-orbital injection of ASCs had no effect on neuropathic pain behaviors. These results suggest a novel and alternative therapeutic application of ASCs to target specific neuropathic pain behaviors.

Effect of two-week continuous epidural administration of 2% lidocaine on mechanical allodynia induced by spinal nerve ligation in rats.

BackgroundLidocaine is an effective against certain types of neuropathic pain. This study aimed to investigate whether timing of initiating continuous epidural infusion of lidocaine affected the glial activation and development of neuropathic pain induced by L5/6 spinal nerve ligation (SNL) in rats.MethodsFollowing L5/6 SNL, rats were epidurally infused 2% lidocaine (drug infusion initiated on days 1, and 7 post SNL model establishment) or saline (saline infusion initiated on day 1 post SNL model establishment) continuously for 14 days. Mechanical allodynia of the hind paw to von Frey filament stimuli was determined prior to surgery, postoperative day 3, and once weekly after SNL model establishment. At 7 days after the infusion of saline or lidocaine ended, spinal activation of proinflammatory cytokines and astrocytes was evaluated immunohistochemically, using antibodies to interleukin-6 (IL-6) and glial fibrillary acidic protein (GFAP).ResultsContinuous epidural administration of 2% lidocaine for 14 days increased the mechanical withdrawal threshold regardless of the difference in timing of initiating lidocaine administration. Epidurally infusing 2% lidocaine inhibited nerve ligation-induced IL-6 and GFAP activation. In the 2% lidocaine infusion group, rats maintained the increased mechanical withdrawal threshold even at 7 days after the discontinuation of 2% lidocaine infusion. ConclusionsContinuous epidural administration of 2% lidocaine inhibited the development of SNL-induced mechanical allodynia and suppressed IL-6 and GFAP activation regardless of the difference in timing of initiating lidocaine administration.

Astrocytic NDRG2 is critical in the maintenance of neuropathic pain

Activation of astrocytes and abnormal synaptic glutamate metabolism are closely associated with the induction and maintenance of neuropathic pain (NP), but the exact mechanism underlying this association remains unclear. N-myc downstream-regulated gene 2 (NDRG2), a novel tumor-suppressor protein and stress-response gene, is involved in the pathogenesis of several neurodegenerative diseases. However, its role in nociceptive transduction has rarely been investigated. Here, we found that NDRG2, which was mainly expressed in the astrocytes in the central nervous system (CNS), was increased in the spinal cord of a spinal nerve ligation (SNL) rat model for NP. Suppression of NDRG2 by intrathecal injection of an NDRG2-RNAi-adenovirus significantly alleviated SNL-induced mechanical and thermal hypersensitivity, as well as elevated astrocytic glutamate transporter 1 (GLT-1) expression and downregulated pro-inflammatory cytokine levels, in the spinal dorsal horn of rats on Day 10 after SNL. Furthermore, in lipopolysaccharide (LPS)-stimulated primary astrocytic cultures derived from neonatal rats, inhibition of NDRG2 significantly reversed both the LPS-induced activation of astrocytes and decreased expression of GLT-1. By contrast, overexpression of NDRG2 by an adenoviral vector carrying NDRG2 resulted in astrocytic activation, aberrant glutamatergic neurotransmission, and spontaneous nociceptive responses in rats. Intrathecal injection of AG490, which is an inhibitor of the Janus tyrosine kinase and signal transducer and activator of the transcription 3 (JAK/STAT3) signaling pathway, significantly attenuated both mechanical and thermal hyperalgesia, as well as inhibited reactive astrocytes and restored normal expression levels of astrocytic GLT-1, in the spinal dorsal horn of NDRG2-overexpression rats. In conclusion, spinal astrocytic NDRG2 is critical in the maintenance of NP. Moreover, NDRG2 modulates astrocytic activation and inflammatory responses via regulating GLT-1 expression through the JAK/STAT3 signaling pathway. Our findings suggested that NDRG2 could be a novel therapeutic target for the treatment of NP.