Paw Withdrawal Latency Values Research Articles

Suberoylanilide Hydroxamic Acid Ameliorates Pain Sensitization in Central Neuropathic Pain After Spinal Cord Injury via the HDAC5/NEDD4/SCN9A Axis.

Pain sensitization in spinal cord injury (SCI)-induced central neuropathic pain has been a research target. Additionally, suberoylanilide hydroxamic acid (SAHA) has been reported to protect against pain hypersensitivity in central neuropathic pain. Hence, this research probed the impact of SAHA on pain sensitization in central neuropathic pain after SCI via the HDAC5/NEDD4/SCN9A axis. After SAHA treatment, SCI modeling, and gain- and loss-of-function assays, behavioral analysis was performed in mice to evaluate pain hypersensitivity and anxiety/depression-like behaviors. The enrichment of H3K27Ac in the NEDD4 promoter and the ubiquitination of SCN9A were measured with ChIP and Co-IP assays, respectively. The treatment of SAHA regained paw withdrawal threshold and paw withdrawal latency values, entry time and numbers in the center area, and entry proportion in the open arm for SCI mice, accompanied by decreases in immobility time, eating latency, thermal hyperalgesia, and mechanical ectopic pain. However, SAHA treatment did not affect the motor function of mice. SAHA treatment lowered HDAC5 expression and SCN9A protein expression in SCI mice, as well as enhanced SCN9A ubiquitination and NEDD4 expression. HDAC5 knockdown greatly increased H3K27Ac enrichment in the NEDD4 promoter. NEDD4 upregulation or HDAC5 knockdown elevated SCN9A ubiquitination but diminished SCN9A protein expression in dorsal root ganglions of SCI mice. NEDD4 silencing mitigated the improving effects of SAHA treatment on the pain hypersensitivity and anxiety/depression-like behaviors of SCI mice. SAHA suppressed HDAC5 to augment NEDD4 expression and SCN9A degradation, thereby ameliorating the pain hypersensitivity and anxiety/depression-like behaviors of SCI mice.

Nanofat lysate ameliorates pain and cartilage degradation of osteoarthritis through activation of TGF-β-Smad2/3 signaling of chondrocytes.

Introduction: Nanofat is an effective cell therapy for osteoarthritis (OA). However, it has clinical limitations due to its short half-life. We developed Nanofat lysate (NFL) to overcome the defect of Nanofat and explore its anti-OA efficacy and mechanism. Methods: Monoiodoacetate (MIA) was employed to establish rat OA model. For pain assessment, paw withdrawal latency (PWL) and thermal withdrawal latency (TWL) were evaluated. Degeneration of cartilage was observed by histopathological and immunohistochemical examination. Primary chondrocytes were treated with TNF-α to establish the cellular model of OA. MTT, wound healing, and transwell assays were performed to assess effects of NFL on chondrocytes. RNA-seq, qPCR and Western blot assays were conducted to clarify the mechanism of NFL. Results and Discussion: The animal data showed that PWL and TWL values, Mankin's and OARSI scorings, and the Col2 expression in cartilage were significantly improved in the NFL-treated OA rats. The cellular data showed that NFL significantly improved the proliferation, wound healing, and migration of chondrocytes. The molecular data showed that NFL significantly restored the TNF-α-altered anabolic markers (Sox9, Col2 and ACAN) and catabolic markers (IL6 and Mmp13). The RNA-seq identified that TGF-β-Smad2/3 signaling pathway mediated the efficacy of NFL, which was verified by qPCR and Western blot that NFL significantly restored the abnormal expressions of TGFβR2, phosphorylated-Smad2, phosphorylated-Smad2/3, Col2, Mmp13 and Mmp3. After long-term storage, NFL exerted similar effects as its fresh type, indicating its advantage of storability. In sum, NFL was developed as a new therapeutic approach and its anti-OA efficacy and mechanism that mediated by TGF-β-Smad2/3 signaling was determined for the first time. Besides, the storability of NFL provided a substantial advantage than other living cell-based therapies.

Evaluation of the paw withdrawal latency for the comparison between tramadol and butorphanol administered locally, in the plantar surface of rat, preliminary study.

The aim of the study was to evaluate the analgesic efficacy of tramadol compared to butorphanol administered locally in ventral surface of the hind paw of rats. Prospective, randomized experimental study; twenty-one adult male Wistar rats were selected. Heart rate (beats minute-1), respiratory rate (breaths minute-1), and paw withdrawal latency (onset of radiant heat until paw withdrawal/seconds) were measured prior (T0) and after (T5, T10, T15, T20) intraplantar injection with saline solution 0,9% (group S), butorphanol 1 mg kg-1 (group B), and tramadol 1 mg kg-1 (group T). Shapiro-Wilk normality test and Friedman test were used to analyze the data expressed by median and range. Statistical significance was set at p < 0.05. Statistical analysis of heart rate showed that there were significant differences between groups at different monitoring times. There were no significant differences in respiratory rate after intraplantar injection in any of the treatment groups. The paw withdrawal latency values at T5, T10, and T15 minutes after intraplantar injection in the group B were significantly higher compared to baseline value and to the values of the other groups. The paw withdrawal latency were no significant changes in the measurements of intragroup in S and T. Intraplantar administration of butorphanol provides a good analgesia and significantly increases paw withdrawal latency compared to tramadol. Intraplantar injection of butorphanol could be useful and safe and safe technique to achieve local analgesia for minor surgical procedures in rats.

Investigation of the role of miR-221 in diabetic peripheral neuropathy and related molecular mechanisms.

Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes, but the molecular mechanisms of DPN are still unclear. To investigate the role of miR-221 in DPN and the related molecular mechanisms. Streptozotocin (STZ) was used to establish an in vivo DPN model. An in vitro DPN model was established using high glucose-induced SH-SY5Y cells. The pain condition of rats was measured by evaluating the 50% paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). Serum exosomes were extracted and identified. Expression of miR-221 in serum exosomes and serum SOCS3 expression were determined using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Western blotting was used to measure the protein levels of SOCS3, bradykinin (BK) and prostaglandin E2 (PEG2). The dual luciferase reporter assay was performed to confirm SOCS3 3'-UTR as a target of miR-221. The serum or cell supernatant levels of PEG2, BK, interleukin (IL)-6, IL-1β, and tumor necrosis factor alpha (TNF-α) were measured using enzyme-linked immunosorbent assay (ELISA). Induction of the lenti-miR-221 inhibitor significantly decreased the expression of miR-221 in DPN rats. Both 50% PWT and PWL values were markedly decreased in DPN rats. When miR-221 was inhibited, the 50% PWT and PWL values were both significantly increased. Knockdown of miR-221 significantly increased the expression of SOCS3 and decreased the expression of NF-κB. Furthermore, knockdown of miR-221 remarkably decreased the expression of PEG2, BK, IL-6, IL-1β, and TNF-α in both STZ-treated DPN rats and high glucose-induced SH-SY5Y cells, which was reversed by inhibition of SOCS3. The dual luciferase reporter assay showed that miR-221 directly targeted and negatively regulated SOCS3. Inhibition of miR-221 can reduce pain and decrease expression of inflammatory factors through targeting SOCS3 in DPN.

Mechanism of Incisional Pain: Novel Finding on Long Noncoding RNA XIST/miR-340-5p/RAB1A Axis

The objective of this study is to investigate the effect of long noncoding RNA (lncRNA) XIST on postoperative pain and inflammation of plantar incision pain (PIP) in rats and its underlying mechanisms.PIP rat models were established by plantar incision. Rats in the sham group were subjected to povidone-iodine scrubbing, and no incision was made. To explore the role of XIST/miR-340-5p/RAB1A in postoperative pain and inflammation, PIP rats were separately or simultaneously injected with lentivirus containing sh-NC, sh-XIST, mimic NC, miR-340-5p mimic, inhibitor NC, miR-340-5p inhibitor, pcDNA3.1, or pcDNA3.1-RAB1A through an intrathecal catheter. The paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) values of rats in each group were assessed to evaluate the pain behavior. RT-qPCR and Western blot were utilized to determine the levels of XIST, miR-340-5p, RAB1A, and NF-κB pathway-related proteins (p-IκBα, IκBα, p-p65, and p65). The concentrations of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in rat spinal dorsal horn tissues were inspected by ELISA. H and E staining was applied to observe the pathological changes of neurons in the spinal dorsal horn, TUNEL staining to detect neuronal apoptosis, and immunohistochemistry to measure RAB1A level.Plantar incision surgery caused decreased PWT and PWL values, enhanced levels of XIST, RAB1A, and inflammatory cytokines, along with an increased proportion of apoptotic neurons. The pain sensitivity and inflammation of rats were motivated after plantar incision surgery. Intrathecal injection of sh-XIST or miR-340-5p mimic ameliorated the pain and inflammation of PIP rats, while silencing of miR-340-5p or overexpression of RAB1A partly reversed the effect of sh-XIST on PIP rats. XIST targeted miR-340-5p and miR-340-5p negatively regulated RAB1A. The XIST/miR-340-5p/RAB1A axis activated the NF-κB signaling pathway.LncRNA XIST aggravates inflammatory response and postoperative pain of PIP rats by activating the NF-κB pathway via the miR-340-5p/RAB1A axis.

Vitexin alleviates neuropathic pain in a mouse chronic constriction injury model by inactivation of NF-κB

Purpose: To investigate the therapeutic effect of vitexin on neuropathic pain (NP) in a mouse model of chronic constriction injury (CCI).Methods: The CCI model was established by four chronic ligatures in the sciatic nerve. Vitexin was intraperitoneally administered (10 mg/kg, once daily) for 21 days. Mechanical withdrawal threshold (MWT) and paw withdrawal latency (PWL) were determined before and after the establishment of CCI model. The spinal cords were collected to measure mRNA levels by reverse-transcriptase polymerase chain reaction (RT-PCR) enzyme-linked immunosorbent assay (ELISA). Western blot was used to examine protein expression levels.Results: Vitexin reversed the CCI-induced reduction in MWT and PWL values, indicating that it lowered mechanical hypersensitivity response and hyperalgesia caused thermal stimulation (p &lt; 0.05). The elevated levels of IL-6, IL-1β, and TNFα observed in CCI-treated mice were also inhibited by vitexin, suggesting that it suppressed pro-inflammatory cytokines. Moreover, vitexin attenuated CCI-induced activation of NF-κB signaling in CCI-treated mice (p &lt; 0.05).Conclusion: Vitexin alleviates NP by inhibiting pro-inflammatory cytokines and NF-κB signaling in CCItreated mice. Thus, it is a potential target for NP treatment.&#x0D; Keywords: Vitexin, Neuropathic pain, Chronic constriction injury, Mechanical hypersensitivity, Hyperalgesia, NF-κB

Adding Dexmedetomidine to Articaine Increases the Latency of Thermal Antinociception in Rats.

Articaine is a low-toxicity local anesthetic that is widely used in dentistry. Typically, epinephrine is added to prolong the duration of articaine local anesthesia; however, epinephrine exhibits adverse effects. Low-dose dexmedetomidine (DEX), an α2-adrenoreceptor agonist, reportedly prolongs local anesthesia without notable adverse cardiovascular effects. The purpose of this study was to assess whether a combination of low-dose DEX and articaine would provide a low-toxicity local anesthetic option for dental procedures without adverse cardiovascular effects. Thus, this study investigated whether DEX could prolong the local anesthetic effect of articaine using a rat model of pain. Adult male Wistar rats (N = 44; 11 per group) received a 50-μL subcutaneous injection into the plantar surface of the hind paws; injections were composed of either normal saline, 4% articaine (2 mg articaine), combined 5 μg/kg DEX and 4% articaine (1.25 μg DEX + 2 mg articaine), or combined epinephrine (1:100,000) and 4% articaine (0.9 μg epinephrine + 2 mg articaine). Subsequent acute pain perception was determined by paw withdrawal movement in response to infrared radiant heat stimulation of the plantar region. Paw withdrawal latency was tested at 5-minute intervals. Paw withdrawal latency values at 35 and 40 minutes were 3.83 ± 1.76 and 3.29 ± 1.43 seconds for articaine alone, 7.89 ± 2.72 and 7.25 ± 3.37 seconds for DEX and articaine, and 8.95 ± 2.28 and 8.17 ± 3.01 seconds for epinephrine and articaine. DEX prolonged the paw withdrawal latency of articaine for up to 35 minutes (p = .015) but not 40 minutes after injection (p = .052) when compared to articaine alone. The combination of DEX and articaine can provide effective local anesthesia for up to 35 minutes after injection.

Intrathecal injection of dexmedetomidine ameliorates chronic neuropathic pain via the modulation of MPK3/ERK1/2 in a mouse model of chronic neuropathic pain

ABSTRACTObjective: Despite the application of dexmedetomidine (DEX) as a perioperative adjuvant in local analgesia, the exact analgesic mechanism underpinning chronic neuropathic pain (CNP) awaits our elucidation.Methods: We investigated the molecular mechanisms of the anti-nociceptive effect of DEX on neuropathic pain in a mouse model induced by chronic constriction injury (CCI).Results: DEX administration significantly increased the paw withdrawal latency (PWL) values 0.5 to 2 h post-injection in CCI-induced CNP mice at day 5 to 21 versus dimethyl sulfoxide (DMSO)-treated mice, confirming its analgesic effect. The c-Fos expression was significantly elevated in CCI mice versus the sham-operated group, whereas the elevation was mitigated by DEX injection. Subsequently, the involvement of MKP1 and MKP3 in the pathogenesis of chronic neuropathic pain was evaluated. Western blotting analyses revealed significant decrease in both MKP1 and MKP3 in the spinal cord in CCI group versus the sham group. DEX markedly elevated the MKP3 expression and modestly reduced the MKP1 expression, with insignificant difference in the latter. Co-injection of BCI (an MKP3 inhibitor) and DEX evidently reduced the PWL values in CCI mice. Furthermore, DEX significantly downregulated the phosphorylation of extracellular-signal-regulated kinase (ERK) 1/2, down-stream effector of MKP3 in CCI mice, whereas the downregulation was reversed by BCI.Conclusion: We confirmed that DEX exerts the analgesic effect on chronic neuropathic pain via the regulation of MKP3/ERK1/2 signaling pathway, which may contribute to clarification of the molecular mechanism and novel therapy for chronic neuropathic pain.

Participation of local exosomes of acupoints in the initiation of acupuncture analgesic effect

ObjectiveTo investigate the local initiation role of local exosomes of acupoints in acupuncture analgesic effect. MethodsThirty-two rats with adjuvant arthritis were randomly divided into model group (Group CFA), model + electroacupuncture group (Group EA), model + antagonist + electroacupuncture group (Group GW4869 + EA), and model + dimethyl sulfoxide + electroacupuncture group (Group DMSO + EA), with 8 rats in each group. The model rat s of adjuvant arthritis were prepared by intradermal injection of 0.1 mL of Freund's adjuvant complete into the metatarsal of the right posterior foot to induce inflammation. No intervention was given in Group CFA, while electroacupuncture was performed in the other three groups at “Zúsānlĭ (足三里 ST 36, bilaterally)” and “Huántiào (环跳 GB 30, bilaterally)” of the rats with the following electroacupuncture parameters: dilatational wave with a frequency of 2/10 Hz, an intensity of 5/10/15 (0.76/1.53/2.3 mA), a duration of 30 min, and an intensity increasing every 10 min. In Group DMSO + EA, DMSO (with a concentration of 0.2%, 50µL/acupoint) was injected at bilateral “ST 36” of the model rats one hour before electroacupuncture, while GW4869 (with a concentration of 3 mg/mL, 50µL/acupoint) was injected at bilateral ST 36 of the model rats one hour before electroacupuncture in Group GW4869 + EA. The paw withdrawal latency (PWL) was used as the therapeutic effect index. ResultsThe PWL of rats in each group at Hour 24 after modeling was significantly lower than that before modeling, indicating that the models were successfully established. After the electroacupuncture treatment, the PWL of rats showed an increasing trend in all groups. The PWL of Group GW4869 + EA (6.74 ± 1.09)s was lower than that of Group EA (7.72 ± 1.54)s on Day 1 after injection, but the difference was not statistically significant (P > 0.05). The PWL values of Group GW4869 + EA (7.72 ± 0.70)s, (7.87 ± 0.58)s were significantly lower than those of Group EA (9.96 ± 0.94)s, (9.66 ± 0.96)s (both P < 0.05) on Days 2 and 3 after injection, and the differences were statistically significant. ConclusionIt was preliminarily found that acupuncture analgesic effect was significantly reduced after local exosomes of acupoints were blocked, indicating that local exosomes of acupoints may be involved in the initiation process of acupuncture effect.

Antinociceptive effects of dezocine on complete Freund's adjuvant-induced inflammatory pain in rats.

Inflammatory pain is known to severely impact the life quality of patients. Notably, dezocine is widely used for the treatment of pain. Therefore, the current study aimed to examine the effects of dezocine on a complete Freund's adjuvant (CFA)-induced inflammatory pain model in rats and to investigate the possible underlying molecular mechanisms. Rats were randomly divided into three groups, including the control, CFA and dezocine+CFA groups, and then subcutaneously injected with 100 µl saline, subcutaneously injected with 100 µl CFA or pretreated with 1 ml dezocine (0.4 µg/kg) at 30 min before CFA injection in the plantar surface of right hind paw, respectively. The paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were measured with a dynamic plantar esthesiometer at 1 day before and 6 h after CFA injection. The ipsilateral lumbar spinal cords of all the rats were harvested for detecting the expression profiles of phosphorylated (p)-p65, p-extracellular signal-regulated kinase 1/2 (p-ERK1/2), cyclooxygenase-2 (COX-2), interleukin (IL)-1β and tumor necrosis factor (TNF)-α by western blot analysis and/or reverse transcription-quantitative polymerase chain reaction. In addition, prostaglandin E2 (PGE2) expression was determined by enzyme-linked immunosorbent assay. Compared with the control group, CFA-induced peripheral inflammation downregulated the PWT and PWL values of rats, which were significantly alleviated by dezocine treatment. Furthermore, the protein levels of p-p65, p-ERK1/2, COX-2, PGE2, IL-1β and TNF-α were significantly upregulated following CFA injection, while they were suppressed by dezocine pretreatment. In conclusion, the analgesic effect of dezocine on inflammatory pain induced by CFA may be associated with the inhibition of the spinal ERK1/2-COX-2 pathway.

The role of p38MAPK activation in spinal dorsal horn in remifentanil-induced postoperative hyperalgesia in rats

Objectives: Remifentanil may induce hyperalgesia. Recent studies implicate a close relationship between post-surgical hyperalgesia and phosphorylation and activation of p38 mitogen-activated protein kinase (p38MAPK) in the spinal microglia. This study aimed to investigate whether the combination of post-surgical and remifentanil-induced hyperalgesia worsens post-operative pain and whether phosphorylated p38MAPK (phospho-p38MAPK) in the spinal dorsal horn in rats is involved in remifentanil-induced postoperative hyperalgesia.Methods: Sprague-Dawley rats were randomly divided into six groups: control, incision only, remifentanil only, remifentanil + incision, remifentanil + incision + SB203580, and remifentanil + incision + DMSO. The p38MAPK inhibitor SB203580 and DMSO were injected intrathecally. A right plantar surgical incision was performed in the incision groups, and remifentanil was infused for 60 min in the remifentanil groups. Mechanical paw withdrawal threshold (PWT) and thermal paw withdrawal latency (PWL) of the bilateral hind paws were measured and the number of phospho-p38MAPK-positive cells in rat spinal dorsal horn sections was counted.Results: Intravenous remifentanil infusion decreased bilateral plantar PWL values from 1 h to 3 days after surgery, however there was no additive effect with incision-induced values. There was a significant increase in the number of dorsal horn phospho-p38MAPK-positive cells in the remifentanil + incision group compared to the incision group, but no increase in the number of these cells when remifentanil was given alone. Intrathecal pretreatment with SB203580 attenuated remifentanil + incision–induced postoperative hyperalgesia and significantly reduced activation of phospho-p38MAPK in spinal dorsal horn.Conclusions: Incision-induced and remifentanil-induced increases in hyperalgesia were not additive when incision and remifentanil were used together. Data on phospho-38MAPK activation in remifenanil-induced hyperalgesia were contradictory and need further clarification.

Minocycline attenuates pain by inhibiting spinal microglia activation in diabetic rats.

The mechanisms associated with diabetes-induced neuropathic pain are complex and poorly understood. In order to understand the involvement of spinal microglia activity in diabetic pain, the present study investigated whether minocycline treatment is able to attenuate diabetic pain using a rat model. Diabetes was induced using a single intraperitoneal injection of streptozotocin (STZ). Minocycline was then intrathecally administered to the rats. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were tested weekly. The expression of OX-42, Iba-1, phospho-p38 mitogen-activated protein kinase (MAPK), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS), were examined in the spinal cord in order to evaluate the activation of microglia. The present study demonstrated that rats with STZ-induced diabetes exhibited increased mean plasma glucose concentration, decreased mean body weight and significant pain hypersensitivity compared with control rats. PWT and PWL values of rats with STZ-induced diabetes increased following treatment with minocycline. No differences were observed in expression levels of the microglial activity markers (OX-42, Iba-1 and phospho-p38 MAPK) between rats with STZ-induced diabetes and control rats. However, TNF-α, IL-1β and iNOS expression levels were higher in rats with STZ-induced diabetes compared with control rats. Following treatment with minocycline markers of microglial activation, including cytokines and iNOS, were downregulated in rats with STZ-induced diabetes. The results of the present study indicated that minocycline treatment may inhibit spinal microglial activation and attenuate diabetic pain in rats with STZ-induced diabetes.

Intravenous Mononuclear Marrow Cells Reverse Neuropathic Pain from Experimental Mononeuropathy

Stem cells mediate neuroprotection in a variety of nervous system injury models. In this study, we evaluated a potential role for stem cells in pain therapies. Marrow mononuclear cells containing mixed stem cell populations were used because of wide experience with these cells in experimental and clinical transplantation. After sciatic nerve chronic constriction injury (CCI), adult male Sprague Dawley rats were treated with freshly isolated marrow mononuclear cells (10(7) cells in 0.5 mL IV) from the same strain, or with carrier. The major end points of analysis were thermal and mechanical hypersensitivity using paw withdrawal latency (PWL) to a calibrated heat source and paw withdrawal response to von Frey filaments, evaluated by a blinded investigator. Marrow transplantation did not prevent pain, and 5 days after CCI all animals were equivalently lesioned. However, 10 days after CCI, rats that received marrow transplants demonstrated paw withdrawal response and PWL patterns indicating recovery from pain, whereas untreated rats continued to have significant pain behavior patterns. For example, PWL values for marrow-treated animals were similar to baseline pre-CCI values (P = 0.54) but significantly shorter latency to withdrawal indicative of continuing pain was seen in untreated rats compared with pre-CCI values (P < 0.001). These studies suggest that stem or progenitor cell-mediated therapies may be useful for the treatment of pain after nerve injury, and deserve further study to elucidate the mechanisms of analgesia.

Effects of gender and gonadal hormones on nociceptive responses to intraplantar carrageenan in the rat

The effects of gender and gonadal hormones on nociceptive responses to intraplantar carrageenan in the rat were investigated. A plantar analgesic meter was used to measure carrageenan-induced changes in paw withdrawal latency (PWL) values, and von Frey monofilaments were used to assess changes in paw withdrawal threshold (PWT) values in response to tactile-evoked (mechanical) stimuli. The data revealed that PWL values were significantly greater in gonadally-intact females than in gonadally-intact males, and this response was abolished by surgical gonadectomies. Gonadally-intact as well as gonadectomized male and female rats also exhibited significant carrageenan-induced decreases in PWT, but neither sex- nor gonadectomy-related differences were detected in the development of mechanical allodynia. These findings demonstrate that intraplantar carrageenan induces nociceptive behaviors in rats that are differentially affected by sex and gonadal hormones.