Nrf2/HO-1-sulfiredoxin1 pathway involved in nanobubble hydrogen-dissolved water-mediated protective effects by ultrasound-guided early local injection in a rat model of chronic constriction injury-induced neuropathic pain

Objective To investigate the effects of intrathecal injection (IT) of agonist and antagonist of N-methyl-D-aspartate receptor subunit 1 (NMDA NR1) on pain threshold and spinal levels of glutamate and glutamate transporter-1(GLT-1) in rat model of chronic constriction injury (CCI) induced neuropathic pain. Methods Sprague-Dawley (SD) rats were randomly divided into 4 groups (Sham, CCI, NMDA, and Humanin). The CCI model was established by right sciatic nerve constriction. The thermal and mechanical thresholds were assessed by paw withdrawal latency (PWL) to radiant heat and von Frey filaments at 1, 3, 5, 7, 10 and 14 days as well as behavior after CCI. The NMDA and Humanin groups were administered intrathecally for 3 days before surgery. Spinal expression of GLT-1 assessed by Weston blotting and levels of glutamate were measured by enzyme-linked immunosorbent assay (ELISA). Results Compared to Sham group, the rats in CCI group gradually appeared the symptoms of toe close together, foot valgus and repeated licking to the operative side of the hind legs after surgery, and the paw withdrawal thresh-old (PWT), and PWL were greatly decreased, reaching the lowest level on the 7th day. Meanwhile, the glutamate content was increased, and the GLT-1 expression was decreased in spinal cord (P<0.05). Compared to CCI group, the rats in NMDA group were observed with further decrease in PWT, PWL and GLT-1 expressions at all observed timepoints (P<0.05), but increase in glutamate content in spine cord (P<0.05). Compared to CCI group, the rats in Humanin group were observed with increase in PWT, PWL and GLT-1 expressions at all observed timepoint (P<0.05), but decrease in glutamate content in spine cord (P<0.05). Conclusions NMDA receptor NR1 subunit participated in regulating neuropathic pain, inhibiting NMDA receptor NR1 subunit can alleviate neuropathic pain by down-regulating of glutamate and GLT-1. Key words: Receptors, N-methyl-D-aspartate/AG/AI/ME; Neuralgia/ME/DT; Glutamic acid/ME/DE; Spinal cord/ME

BackgroundNeuropathic pain belongs to chronic pain and is caused by the primary dysfunction of the somatosensory nervous system. Long noncoding RNAs (lncRNAs) have been reported to regulate neuronal functions and play significant roles in neuropathic pain. DLEU1 has been indicated to have close relationship with neuropathic pain. Therefore, our study focused on the significant role of DLEU1 in neuropathic pain rat models.MethodsWe first constructed a chronic constrictive injury (CCI) rat model. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were employed to evaluate hypersensitivity in neuropathic pain. RT-qPCR was performed to analyze the expression of target genes. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect the concentrations of interleukin‐6 (IL-6), tumor necrosis factor‐α (TNF-α) and IL-1β. The underlying mechanisms of DLEU1 were investigated using western blot and luciferase reporter assays.ResultsOur findings showed that DLEU1 was upregulated in CCI rats. DLEU1 knockdown reduced the concentrations of IL‐6, IL‐1β and TNF‐α in CCI rats, suggesting that neuroinflammation was inhibited by DLEU1 knockdown. Besides, knockdown of DLEU1 inhibited neuropathic pain behaviors. Moreover, it was confirmed that DLEU1 bound with miR-133a-3p and negatively regulated its expression. SRPK1 was the downstream target of miR-133a-3p. DLEU1 competitively bound with miR-133a-3p to upregulate SRPK1. Finally, rescue assays revealed that SRPK1 overexpression rescued the suppressive effects of silenced DLEU1 on hypersensitivity in neuropathic pain and inflammation of spinal cord in CCI rats.ConclusionDLEU1 regulated inflammation of the spinal cord and mediated hypersensitivity in neuropathic pain in CCI rats by binding with miR-133a-3p to upregulate SRPK1 expression.

Purpose: To investigate the effect of miR-665 in neuropathic pain and the possible molecular mechanism involved.Methods: A neuropathic pain model was established using chronic constriction injury (CCI) methods in Sprague Dawley (SD) rats. Mechanical and thermal hyperalgesia were measured using paw withdrawal threshold (PWT) and paw withdrawal latency (PWL), respectively. The inflammation response was determined by assessing the production of inflammation factors. The target relationship of miR-665 and suppressor of cytokine signaling 1 (SOCS1) was verified by luciferase assay.Results: In the CCI rat model, PWT and PWL decreased following treatment with miR-665 (p &lt; 0.01). MiR-665 was elevated in the spinal cord and microglia of CCI rats at different time points (p &lt; 0.01). Down-regulation of miR-665 increased PWT and PWL and inhibited the production of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in CCI rats (p &lt; 0.01). Luciferase assay results indicate that SOCS1 was the target of miR-665 (p &lt; 0.01). SOCS1 decreased in CCI rats (p &lt; 0.01) after treatment with miR-665. MiR-665 negatively regulated the expression of SOCS1 (p &lt; 0.01). Down-regulation of SOCS1 reversed the alleviating effect of decreased miR-665 on pain sensitivity and inflammationresponse (p &lt; 0.01).Conclusion: Down-regulation of miR-665 alleviates neuropathic pain by targeting SOCS1, and hence making miR-665 a promising therapeutic target for neuropathic pain.&#x0D; Keywords: MiR-665, SOCS1, Neuropathic pain, CCI, Spinal cord

Objective To observe the effect of early use of 0.25 MPa hyperbaric oxygen (HBO) on pain behavior in model rat of neuropathic pain (NP) and explore the mechanism. Methods Fifty-four male Sprague⁃Dawley (SD) rats were randomly divided into 3 groups (18 rats per group) including sham operation, sciatic nerve ligation with chronic constriction injury (CCI) and HBO early⁃treatment groups. The rat model of neuropathic pain was established, and CCI + HBO group received 0.25 MPa HBO treatment for 60 min per day at early stage, continuing for 5 d. The changes in general state of health, the number of paw withdrawal responses, paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were assessed at different time points after operation. Results Compared with sham-operated group, the body weight of rats in CCI group decreased significantly (t = 4.772, P = 0.000) on the 14th day; the body weight of rats in CCI + HBO group was significantly higher than that in CCI group (t = 2.411, P = 0.029). Compared with sham-operated group, reduced PWL immediately after operation (t = 28.345, P = 0.000), and more paw withdrawal responses (t = 12.541, P = 0.000) and decreased PWT (t = 4.032, P = 0.001) from the 3rd day after operation were observed in CCI group. Compared with CCI group, the number of paw withdrawal responses decreased (t = 8.077, P = 0.000), and PWT and PWL increased (t = 2.114, P = 0.049; t = 7.715, P = 0.000) in CCI + HBO group. Conclusion 0.25 MPa HBO treatment at early stage can ameliorate the mechanical and thermal hyperalgesia. HBO may provide a new convenient and effective way for the treatment of neuropathic pain in clinic.

Valproate (VPA) is a well-known drug for treating epilepsy and mania, but its action in neuropathic pain is unclear. We used a chronic constriction injury (CCI) model to explore whether VPA prevents neuropathic pain-mediated inflammation and neuronal death. Rats were treated with or without VPA. CCI + VPA rats were intraperitoneally injected with VPA (300 mg/kg/day) from postoperative day (POD) 1 to 14. We measured paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) 1 day before surgery and 1, 3, 7, 14 days after CCI and harvested the sciatic nerves (SN), spinal cord (SC) and dorsal root ganglia (DRG) on POD 3, 7, and 14. PWL and PWT were reduced in CCI rats, but increased in CCI + VPA rats on POD 7 and POD 14. VPA lowered CCI-induced inflammatory proteins (pNFκB, iNOS and COX-2), pro-apoptotic proteins (pAKT/AKT and pGSK-3β/GSK-3β), proinflammatory cytokines (TNF-α and IL-1β) and nuclear pNFκB activation in the SN, DRG and SC in CCI rats. COX-2 and pGSK-3 proteins were decreased by VPA on immunofluorescence analysis. VPA attenuated CCI-induced thermal and mechanical pain behaviors in rats in correlation with anti-neuroinflammation action involving reduction of pNFκB/iNOS/COX-2 activation and inhibition of pAKT/pGSK-3β-mediated neuronal death from injury to peripheral nerves.

To explore the analgesic effects of electroacupuncture (EA) and its impact on the EphBs-p38 mitogen-activated protein kinase (MAPK) pathway and microglia in a rat model of neuropathic pain (NP). Following adaptive training, 60 male Sprague Dawley (SD) rats were allocated to one of two experiments. In experiment 1, rats received intrathecal SB203580 (p38 MAPK inhibitor), intramuscular EphB1-Fc (EphBs inhibitor) or no injection before undergoing chronic constrictive injury (CCI). In experiment 2, CCI model rats received EA either alone or combined with either anisomycin (p38 MAPK agonist) or EphrinB1-Fc (EphBs agonist) versus minimal acupuncture (MA) as a control intervention. A sham surgery group was included in both experiments as a control for CCI. All groups consisted of n = 6 rats (four in experiment 1 and six in experiment 2). Behavioral hyperalgesia was examined and the spinal L5-6 region was harvested and subjected to enzyme-linked immunosorbent assay to assess tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels. Western blotting and immunofluorescence were used to assess protein expression of B-cell lymphoma (Bcl)-2, Bcl-2 associated X-protein (BAX), EphB1, EphrinB1, p38 MAPK, phosphorylated (p)-p38 MAPK and ionized calcium binding adaptor molecule (Iba)-1. CCI induced behavioral hyperalgesia, as demonstrated by altered paw withdrawal latency (PWL), paw withdrawal threshold (PWT) and cytokine levels, and increased p38 MAPK phosphorylation and microglial activation. However, inhibitors SB203580 and EphB1-Fc reversed these effects. Notably, EA showed similar beneficial effects, but these were counteracted when combined with anisomycin and EphrinB1-Fc. The analgesic effects of EA in this rat model of NP appear to be linked to diminished p-p38 MAPK expression and subsequent microglial deactivation. EA has a potential role as a complementary therapy for NP.

miR-101 down-regulates mTOR expression and attenuates neuropathic pain in chronic constriction injury rat models

MG53 ameliorates nerve injury induced neuropathic pain through the regulation of Nrf2/HO-1 signaling in rats

This study aimed to determine the role of dexmedetomidine (Dex) in neuropathic pain (NP) after chronic constriction injury (CCI) in a rat model as well as its underlying mechanism. First, a CCI rat model was established. After treatment with Dex, the severity of NP was ascertained by monitoring paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) at different time points. Immunohistochemical analysis was performed to determine the levels of Keap1 and Nrf2 in the spinal cord. Furthermore, the levels of Keap1–Nrf2–HO-1 pathway molecules, apoptotic proteins, and antioxidant genes in the spinal cord or isolated primary microglia were determined using quantitative polymerase chain reaction and western blotting. The release of proinflammatory cytokines was detected via enzyme-linked immunosorbent assay. To evaluate Dex-treated CCI-induced NP via the Keap1–Nrf2–HO-1 pathway, the rats were intrathecally injected with lentivirus to upregulate or downregulate the expression of Keap1. We found that Dex inhibited pathological changes and alleviated sciatic nerve pain as well as repressed inflammation, apoptosis, and redox disorders of the spinal cord in CCI rats. Keap1 protein expression was substantially downregulated, whereas Nrf2 and HO-1 expressions were significantly upregulated in the spinal cord after Dex administration. Additionally, Keap1 overexpression counteracted Dex-mediated inhibition of NP. Keap1 overexpression led to a decrease in Nrf2 and HO-1 levels as well as PWT and PWL but led to an aggravation of inflammation and antioxidant disorders and increased apoptosis. Keap1 silencing alleviated NP in rats with CCI, as evidenced by an increase in PWT and PWL. Keap1 depletion resulted in the alleviation of inflammation and spinal cord tissue injury in CCI rats. Collectively, these findings suggest that Dex inhibits the Keap1–Nrf2–HO-1-related antioxidant response, inflammation, and apoptosis, thereby alleviating NP in CCI rats.

Systemic gabapentin is a mainstay treatment for neuropathic pain though there are side-effects. Localized therapy may curtail such side-effects so a topical gabapentin dermal application was examined in the chronic constriction injury (CCI) model of neuropathic pain. Partial denervation CCI was achieved by rat sciatic nerve ligation. Gabapentin gel (10% w/w) was applied three times daily on the ipsilateral or contralateral plantar surface of the hind-paw, whereas in a concurrent systemic study, gabapentin was intraperitoneally administered daily (75mg/kg) for 30days. Tests for static- and dynamic-mechano-allodynia [paw withdrawal threshold (PWT) to von Frey filament application and latency (PWL) to light brushing], cold-allodynia [paw withdrawal duration (PWD) to acetone], heat- (PWL and PWD) and mechano-hyperalgesia (PWD to pin prick) were utilized to assess pain, whereas effects on locomotion (open field) and motor balance (rotarod and footprint analysis) were measured on days 5-30 post surgery. Topical application of gabapentin gel ipsilaterally but not contralaterally alleviated CCI-induced static- (days 10-30) and dynamic-allodynia (days 15-30), suppressed cold-allodynia (days 10-30), heat- (days 15-30) and mechano-hyperalgesia (days 5-30) indicating a local action. Systemic gabapentin exhibited similar pain profiles but was associated with motor impairment. The gabapentin gel formulation afforded desirable neuropathic pain alleviating effects devoid of unwanted systemic side-effects. These outcomes disclose an expedient pharmacological validation of the effectiveness of topical gabapentin gel against an extensive range of nociceptive stimulus modalities utilizing the CCI-induced neuropathic pain model. They also advocate further clinical studies on topical gabapentin with regard to certain neuropathic pain syndromes. Systemic gabapentin neuropathic pain management carries side-effects ostensibly preventable by localized therapy. This study validates the effectiveness potential of a topical gabapentin gel against an extensive range of nociceptive stimulus modalities utilizing the chronic constriction injury-induced neuropathic pain model.

PurposeThe pathogenesis of neuropathic pain is complex, and previous studies have found that microRNAs are important regulators of neuropathic pain and are associated with the progression of neuropathic pain. This study aims to explore the level and role of miR-204-5p in the chronic constrictive injury (CCI) model of rats.Patients and MethodsThe CCI rat model was constructed to evaluate paw withdrawal threshold (PWT), paw withdrawal latency (PWL), the expressions of miR-204-5p, and the contents of inflammatory factors in the model. Overexpression of miR-204-5p in rat spinal cord was induced by intrathecal injection of miR-204-5p mimics. PWT and PWL were used to estimate mechanical and thermal pain thresholds. IL-6 and TNF-α were determined by ELISA. Luciferase reporter gene was conducted to verify the targeting relationship between miR-204-5p and BRD4.ResultsmiR-204-5p was abnormally down-regulated in the CCI group. The thresholds of mechanical and thermal pain stimulation in the CCI group were lower, and the levels of inflammatory factors were higher than those in the sham group. Overexpression of miR-204-5p alleviated PWT, PWL and inflammatory factors. Besides, the luciferase reporter gene showed that BRD4 was a target gene of miR-204-5p.ConclusionThese results suggested that miR-204-5p may alleviate neuropathic pain and inflammation through targeted regulation of BRD4 expression.

Treatment of neuropathic pain is far from satisfactory. This study sought evidence of a neuroprotective effect of alpha-lipoic acid (ALA) to treat neuropathic pain in a chronic constriction injury (CCI) rat model. A total of 48 rats were randomly divided into sham, CCI, or CCI + ALA groups. Mechanical and thermal nociceptive thresholds were evaluated as behavioral assessments. Dorsal root ganglia cells were assessed morphologically with hematoxylin and eosin staining and for apoptosis with P53 immunohistochemical staining. Compared with the sham group, the CCI group had a shorter paw withdrawal threshold and paw withdrawal latency, abnormal morphologic manifestations, and increased numbers of satellite glial cells and P53+ cells. These changes were significantly reversed by treatment with ALA. Our study indicates neuroprotective effects of ALA on chronic neuropathic pain in a CCI rat model. ALA is potentially considered to be developed as a treatment for neuropathic pain caused by peripheral nerve injury, which requires further verification.

IntroductionNeuropathic pain is pretty common in modern society, and the treatment effect is far from satisfactory. This study aimed to find evidence of the neuroprotective effect of erythropoietin (EPO) in the treatment of neuropathic pain in a rat model of chronic constriction injury (CCI).MethodsA total of 30 rats were randomly divided into sham operation group, CCI group, or CCI+EPO group. The mechanical and thermal nociception thresholds are evaluated as behavioral assessments. The dorsal root ganglion cells were morphologically evaluated by hematoxylin and eosin staining, and AMPK, p-AMPK, mTOR, p70S6K, and AQP-2 proteins were compared and analyzed by Western blotting. Compared with the sham operation group, rats in the CCI group had shorter paw withdrawal threshold and paw withdrawal latency, abnormal morphology, and increased satellite glial cells.ResultsAfter treatment with EPO, these changes were significantly reversed. In vivo administration of erythropoietin seems to be able to regulate the expression of AQP-2 through the AMPK/mTOR/p70S6K pathway. Our study provides behavioral, morphological, and immunoblot evidence to prove the neuroprotective effect of EPO in the treatment of chronic neuropathic pain in the CCI rat model.ConclusionOur results indicate that EPO has the potential to treat neuropathic pain caused by peripheral nerve injury, although further verification is needed.

Objective To investigate the role of cyclooxygenases (COXs) in the up-regulation of the expression of P2X3 receptors in the dorsal root ganglion (DRG) in rats with neuropsthic pain. Methods Twenty-four male SD rats, weighing 250-280 g, were randomly divided into 4 groups ( n = 6 each): sham operation group (group S), chronic constrictive injury (CCI) group, COX-1 inhibitor ibuprofen group (group Ⅰ), and COX-2 inhibitor celecoxib group (group C). Neuropathic pain was induced by CCI. The animals were anesthetized with intraperitoneal 10% chloral hydrate 300-500 mg/kg. CCI was produced by placing 4 ligatures on the left sciatic nerve at 1 mm intervals. In group S, the left sciatic nerve was only exposed but not ligated. In groups Ⅰ and C, ibuprofen 40 mg·kg-1 ·d-1 and celecoxib 30 mg·kg-1 ·d-1 were given through a gastric tube into the stomach at day 3-14 after operation respectively. Paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were measured before operation (baseline), and at 3, 5, 7, 10 and 14 days after operation. Then the rats were sacrificed and their L()-6 DRGs were removed to detect the expression of P2X3 mRNA and protein. Results Compared with group S, PWL was significantly shortened, PWT decreased, and P2X3 mRNA and protein expression up-regulated in group CCI ( P ＜ 0.05＝. Compared with group CCI, PWL was significantly prolonged, PWT increased, and P2X3 mRNA and protein expression down-regulated in groups Ⅰ and C (P ＜0.05＝. Compared with group Ⅰ, PWL was significantly prolonged, PWT increased, and P2X3 mRNA and protein expression up-regulated in group C ( P ＜0.05＝. Conclusion COXs are involved in the up-regulation of the expression of P2X3 receptors in the DRG in rats with neuropathic pain, and the effect of COX-1 is stronger than that of COX-2. Key words: Prostaglandin-endoperoxide synthases; Neuralgia; Receptors, purinergic P2; Ganglia,spinal

Objective To investigate the effect of tramadol on the expression of 5-HT1A receptor in the distal'cerebrospinal fluid contacting neurons (CSF-CNs) in mid-brain in a rat model of neuropathic pain. Methods Forty male SPF SD rats weighing 220-280 g were randomly divided into 5 groups (n = 8 each): group Ⅰ normal control (group C); group Ⅱ normal saline (group NS); group Ⅲ tramodol (group T); group Ⅳ neuropathic pain + normal saline (group NP+ NS) and group Ⅴ neuropathic pain + tramadol (group NP + T). Neuropathic pain was induced by chronic constrictive injury (CCI) in group Ⅳ and Ⅴ . Four silk ligatures were placed on the sciatic nerve at 1 mm intervals. In group Ⅱ (NS) and group Ⅲ (T) the sciatic nerve was exposed but not ligated and NS 2 ml/kg and tramadol 10 mg/kg were injected IP respectively, while in group Ⅳ and Ⅴ NS 2 ml/kg and tramadol 10 mg/kg were injected IP respectively on the 7th day after CCI. Paw withdrawal threshold (PWT) to von Frey filament stimulation and paw withdrawal latency (PWL) to noxious thermal stimuli were measured before (T1) and after IP NS or tramadol injection (T2) in group Ⅱ-Ⅴ. The distal CSF-CNs in the mid-brain was labelled with 30% cholera toxin subunit B and horseradish peroxidase compound (CB-HRP) 3 μl injected in left lateral cerebral ventricle. The expression of 5-HT1A receptors was measured by immuno-histochemistry. Results PWT and PWL were significantly decreased after CCI in group Ⅳ (NP + NS) and tramadol significantly inhibited the mechanical and thermal hyperalgesia in group Ⅴ (NP + T). There was no significant difference in the number of distal CSF-CNs among the 5 groups. CCI significantly down-regulated the expression of 5-HT1A in distal CSF-CNs in group Ⅳ(NP+ NS) as compared with group Ⅰ , Ⅱ and Ⅲ and tramadol significantly inhibited the CCI-induced downregulation of 5-HT1A receptor expression. Conclusion Tramadol can ease neuropathic pain by down-regulating the expression of 5-HT1A receptor in distal CSF-CNs in mid-brain. Key words: Tramadol; Neurons; Receptor, serotonin, 5-HT1A; Neuralgia