Paw Withdrawal Threshold Tests Research Articles

Consecutive assessment of recovery after peripheral nerve injury of the sciatic nerve within the same rat using PET/MRI.

Positron emission tomography (PET) has been reported as effective in diagnosing peripheral nerve injury (PNI). However, there is a lack of studies evaluating different degrees of PNI using PET within the same individual to reduce errors due to interindividual differences. To evaluate the recovery process in the same rat after sciatic nerve injury using PET/magnetic resonance imaging (MRI). Crushing nerve injuries were induced in the left sciatic nerves of six male rats, preserving the right ones. The degree of nerve damage was measured at one, two, three, four, and five weeks postoperatively using three assessment methods: paw withdrawal threshold test (RevWT); PET (SUVR); and MRI (MRSIR). All the representing values of each method are presented as ratio values of the right and left sides in each rat. Significant gradual recovery of all rats was observed over time in all the methods. No significant differences in RevWT and MRSIR were observed between before and more than four weeks after injury, whereas a significant difference in SUVR was still observed between before and five weeks after injury (P = 0.0007). The parameters of all methods decreased significantly over time (P = 0.000, all), and the explanatory power was significant in RevWT, SUVR, and MRSIR. PET and MRI could be valuable non-invasive techniques for diagnosing neuropathic pain resulting from PNI. PET/MRI would be expected to be a more accurate and informative diagnostic tool for PNI than MRI alone.

Evaluation of peripheral nerve injury according to the severity of damage using 18F-FDG PET/MRI in a rat Model of sciatic nerve injury

ABSTRACT Objectives We ascertained that the PET scan may be a valuable imaging modality for the noninvasive, objective diagnosis of neuropathic pain caused by peripheral nerve injury through the previous study. This study aimed to assess peripheral nerve damage according to severity using18F-FDG PET/MRI of the rat sciatic nerve. Methods Eighteen rats were divided into three groups: 30-second (G1), 2-minute (G2), and 5-minute (G3) crushing injuries. The severity of nerve damage was measured in the third week after the crushing injury using three methods: the paw withdrawal threshold test (RevWT), standardized uptake values on PET (SUVR), and intensity analysis on immunohistochemistry (IntR). Results There were significant differences between G1 and G3 in both SUVR and IntR (p = 0.012 and 0.029, respectively), and no significant differences in RevWT among the three groups (p = 0.438). There was a significant difference in SUVR (p = 0.012), but no significant difference in IntR between G1 and G2 (p = 0.202). There was no significant difference between G2 and G3 in SUVR and IntR (p = 0.810 and 0.544, respectively). Discussion Although PET did not show results consistent with those of immunohistochemistry in all respects, this study demonstrated that PET uptake tended to increase with severe nerve damage. If this research is supplemented by further experiments, PET/MRI can be used as an effective diagnostic modality.

Traditional Chinese Manual Therapy (Tuina) Improves Knee Osteoarthritis by Regulating Chondrocyte Autophagy and Apoptosis via the PI3K/AKT/mTOR Pathway: An in vivo Rat Experiment and Machine Learning Study.

Knee osteoarthritis (KOA) is on the rise due to lifestyle changes, obesity, and aging, yet effective treatments are lacking. Traditional Chinese manual therapy (Tuina) is promising for KOA. However, its mechanism remains unclear. This study aims to determine the effects of Tuina on a rat KOA model, focusing on the role of chondrocyte apoptosis and autophagy mechanisms. KOA was induced in rats by intra-articular injection of L-cysteine-activated papain into the right knee. Thirty-six male Sprague Dawley (SD) rats were randomly divided into blank, model control, Tuina, and positive drug groups. Paw withdrawal threshold tests, knee joint swelling, and passive range of motion assessed knee behavior. Cartilage tissue cytology, cytokine contents, and the mRNA and protein expression of PI3K/AKT/mTOR signaling pathway components were analyzed using HE and TUNEL staining, ELISA, RT-qPCR, and Western blotting, respectively. In addition, we used machine learning methods to conduct a secondary analysis of the dataset from the in vivo experiments in rats to verify the findings. Tuina significantly relieved pain and joint swelling, and improved range of motion. Staining showed reduced articular cartilage destruction and apoptosis. Tuina reduced the serum levels of IL-1β, IL-17, MMP-3, and MMP-13. Tuina downregulated Bax, ULK1, Beclin-1, LC3-II/I and upregulated PI3K, AKT, mTOR, and BCL-2 in cartilage tissue. The machine learning results indicated an 83.33% accuracy for the prediction model, remaining stable through both uni- and multivariate analyses. Tuina yielded the best comprehensive efficacy on KOA as well as better rat behavior and PI3K/AKT/mTOR signaling pathway improvement effect than positive drugs, while its cytokine-reducing ability was comparable to that of positive drugs. Tuina can alleviate cartilage tissue injury in KOA, relieve inflammation, and reduce chondrocyte apoptosis and autophagy, the underlying mechanisms of which may be associated with activation of the PI3K/AKT/mTOR signaling pathway.

Antinociceptive and anti-inflammatory activity of DW-1021, the ionic complex of pelubiprofen and tramadol, in rodents

Although drugs such as acetaminophen, opioids, and nonsteroidal anti-inflammatory drugs (NSAIDs), are commonly used for pain management, the side effects of these drugs such as hepatotoxicity, nephrotoxicity, nausea, and vomiting, can not be neglected. Therefore, combinations of analgesics with different mechanisms raise the possibility of developing novel analgesics. Therefore, the aim of the present study was to evaluate whether DW-1021, the ionic complex of pelubiprofen (NSAID) and tramadol (opioid), has synergic antinociceptive and anti-inflammatory effects in nociceptive as well as inflammation-induced nociceptive models compared to pelubiprofen- or tramadol-only administration. Strong synergistic antinociceptive efficacy of DW-1021 was observed in the mouse writhing test and von Frey paw withdrawal threshold test in the carrageenan-induced rats. The hot plate test in mice and the Randall-Selitto mechanical paw pressure test in carrageenan-induced rats revealed that DW-1021 had a preferable effect on relieving pain to pelubiprofen, but not as much as tramadol. In the carrageenan-induced rats, DW-1021 had a more potent effect on reducing paw inflammation (paw volume, width, and thickness) via the suppression of PGE2 production than tramadol, but less than that of pelubiprofen. Taken together, our results suggest that the administration of DW-1021, a combination of pelubiprofen and tramadol, exerted a potent effect and can be used as a potential therapeutic agent for relieving pain and inflammation.

Electroacupuncture ameliorates knee osteoarthritis in rats via inhibiting NLRP3 inflammasome and reducing pyroptosis.

Objective: Knee Osteoarthritis (KOA), is the most common joint disease worldwide. The pathogenesis of KOA is complex and electroacupuncture (EA) is an effective therapy for KOA, but the mechanism remains unclear. In this study, we aim to investigate the potential therapeutic effect of EA on the rat model of KOA induced by monosodium iodoacetate (MIA) and its relationship with NLRP3 inflammasome by immunohistochemistry and western blot. Methods: KOA was induced by intra-articular injection of MIA (3mg/50μL) into the right knee joint of rats. Forty-five male rats weighing 250-300g were randomly divided into 3 groups: control group, KOA group, and KOA + electroacupuncture group (KOA+EA). EA treatment lasted for 2weeks (6 times a week). Paw withdrawal threshold tests were used to assess mechanical allodynia once a week. Safranin O/Fast Green and hematoxylin and eosin (H&E) staining were used to assess the damage to cartilage, synovium, and subpatellar fat pad (IFP). Immunohistochemistry was used to observe NLRP3 inflammasome-associated protein-positive cells in the same field of view and western blot was used to detect the expression of the associated protein in cartilage tissue. Results: The KOA group showed mechanical hyperalgesia, joint inflammation, and significant cartilage tissue destruction. Safranin O/Fast Green and H&E staining revealed that EA alleviated the joint pathological changes caused by KOA and had a protective effect on cartilage, synovium, and IFP destruction. Mechanical allodynia pain and joint swelling were reduced in KOA rats after EA treatment. Immunohistochemistry and western blot showed significant inhibition of NLRP3 inflammasome-associated protein. Conclusion: The results indicate that EA can inhibit NLRP3 inflammasome and reduce pyroptosis, which results in the protection of cartilage tissue and the treatment of KOA. It provides reliable evidence for the development of EA in the treatment of KOA and the clinical application of acupuncture.

Identification of sensory dysfunction and nervous structure changes in Fam134b knockout mice

ABSTRACT Objectives Mutation in human FAM134B gene has been implicated in hereditary sensory and autonomic neuropathy type IIB. We aimed to knock out Fam134b in mice and explored its phenotypes to determine whether the genetic impairments and behavioral changes can mirror manifestations noted in humans. Methods We used CRISPR/Cas9 technology to knockout the Fam134b gene in the C57BL/6 J mouse. After confirming the knockout was successful by Sanger sequencing and Western blot, sensory function was measured using the hot plate test and the 50% paw withdrawal threshold test. In addition, standard microscopy and transmission electron microscopy were performed to observe the structural changes of the dorsal root ganglion sensory neuron and the sciatic nerve. Results DNA sequencing and Western blot analysis confirmed the mutation in the Fam134b mutation gene and the loss of expression of its products. Fam134b knockout mice exhibited heat pain insensitivity and mechanical hyperalgesia. Interestingly, limb damage was found in some homozygotes. Demyelination in the sciatic nerve was common. Golgi bodies were turgid in dorsal root ganglion neuron. Conclusions These findings indicate that peripheral neuropathy is common in Fam134b KO mice. We believe this novel animal model is likely to have significant future potential as a reliable model for the evaluation of peripheral neuropathy and its complications.

Imaging Study on Acupuncture Inhibiting Inflammation and Bone Destruction in Knee Osteoarthritis Induced by Monosodium Iodoacetate in Rat Model

ObjectiveWe aim to explore whether acupuncture inhibits inflammation and bone destruction in rat model monosodium iodoacetate (MIA)-induced knee osteoarthritis (KOA) by 18F-fluorodeoxyglucose (18F-FDG) small-animal positron emission tomography (PET) and micro-computed tomography (CT) imaging.MethodsKOA was induced in rats by intra-articular injection MIA (2 mg/50 μL) through the right knee of the rats. Forty male Sprague Dawley rats weighing 280 to 340 g (12 weeks old) were randomly divided into four groups including Control group, KOA group, KOA plus manual acupuncture group (KOA+MA), KOA plus sham acupuncture group (KOA+SA). The acupuncture treatment lasted for three weeks (one-day rest after six days of treatment). Paw withdrawal threshold test and open-field test were used to assess mechanical allodynia and locomotor activity respectively for once a week. Hematoxylin and eosin (H&E) staining was used to assess the damage of the cartilage, synovium and infrapatellar fat pad (IFP). 18F-FDG PET was performed to quantify joint inflammation. The influence on the subchondral bone in these rats was confirmed by micro-CT.ResultsMechanical hyperalgesia, joint inflammation, and obvious bone destruction were observed in the KOA group. H&E staining of the knee joint found that manual acupuncture played a protective effect in cartilage, synovium and IFP destruction. However, compared with KOA group, the results in sham acupuncture had no significant difference. After manual acupuncture treatment in KOA rats, inflammation was significantly suppressed shown by 18F-FDG PET imaging. Micro-CT analysis of the knee joint revealed that manual acupuncture protected bone by inhibiting osteophyte development and subchondral bone remodeling.ConclusionThe results of 18F-FDG PET and micro-CT showed that manual acupuncture inhibited inflammation and bone destruction, which provides reliable evidence for the effectiveness of acupuncture in hindering development of KOA, and provides reliable evidence for clinical application of acupuncture.

A model of pain behaviors in freely moving rats generated by controllable electrical stimulation of the peripheral nerve

BackgroundNeuropathic pain patients have described experiencing unprovoked, intermittent pain attacks with shooting, stabbing, and burning qualities. Rodent models used in previous literature usually only involve acute exposure, and/or are unable to manipulate the stimulation intensity in vivo by the experimenter during an experiment. New methodThis paper describes a method to induce controllable pain behaviors in rodents using a wireless portable electronic device that can be manipulated within the course of an experiment. A stimulating electrode was implanted at the L5 spinal nerve location in Sprague-Dawley rats and our custom-built wireless stimulating device was attached to deliver variable stimulation in freely moving animals (50 Hz, 0.5 V; 100 Hz, 1 V). ResultsImplantation itself did not induce hypersensitivity as measured by the mechanical paw withdrawal threshold test. Observation of pain behaviors (paw elevation and licking) indicated that high stimulation intensity yielded a significant increase in pain behaviors. Even further, high intensity stimulation resulted in a behavioral “wind-up” of pain behaviors that persisted into the resting period when no stimulation was applied. Comparison with existing methods and conclusionsThis method can be used to study pain behaviors in a controllable way in freely moving rodents in comparison to existing models that are acute and/or are unable to manipulate the stimulation intensity in vivo.

ANTI-NOCICEPTIVE EFFECT OF AGRIMONIA EUPATORIA EXTRACT ON A CISPLATIN-INDUCED NEUROPATHIC MODEL.

Background:Natural products including Agrimonia eupatoria are considered an incomparable source of molecular diversity that has led to the medicines, especially for pain treatment. To investigate the antinociception of Agrimonia eupatoria, we examined its activity in a rat model of cisplatin neuropathy.Materials and Methods:Male Sprague-Dawley rats received intraperitoneal (i.p.) cisplatin twice a week at a dose of 2 mg/kg (cumulative dose, 20 mg/kg) for 4 weeks. Before each injection, 2 ml of sterile saline solution was given subcutaneously to prevent renal damage via hyperhydration. The mice were treated with gabapetin as a positive control drug with a 100mg/kg intraperitoneal injection. A. eupatoria extract of 200mg/kg was solved in saline and then treated by oral administration.Results:The mice treated with A. eupatoria showed lower withdrawal duration in the pin-prick and plantar tests, and a higher withdrawal threshold in the paw-withdrawal threshold test as compared to control animals in a cisplatin-induced neuropathic model. In the case of cold-allodynia, A. eupatoria treatment increased paw-withdrawal duration in a chemical test. A. eupatoria showed a more outstanding effect than gabapentin in all used tests for preventing cisplatin-induced nerve injury for 4 weeks.Conclusions:Our results suggest that A. eupatoria extract showed an antinociceptive effect in the pin-prick test, plantar test, and paw-withdrawal threshold test using a cisplatin-induced neuropathic rat model.

Upregulation of EMMPRIN (OX47) in Rat Dorsal Root Ganglion Contributes to the Development of Mechanical Allodynia after Nerve Injury.

Matrix metalloproteinases (MMPs) are widely implicated in inflammation and tissue remodeling associated with various neurodegenerative diseases and play an important role in nociception and allodynia. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) plays a key regulatory role for MMP activities. However, the role of EMMPRIN in the development of neuropathic pain is not clear. Western blotting, real-time quantitative RT-PCR (qRT-PCR), and immunofluorescence were performed to determine the changes of messenger RNA and protein of EMMPRIN/OX47 and their cellular localization in the rat dorsal root ganglion (DRG) after nerve injury. Paw withdrawal threshold test was examined to evaluate the pain behavior in spinal nerve ligation (SNL) model. The lentivirus containing OX47 shRNA was injected into the DRG one day before SNL. The expression level of both mRNA and protein of OX47 was markedly upregulated in ipsilateral DRG after SNL. OX47 was mainly expressed in the extracellular matrix of DRG. Administration of shRNA targeted against OX47 in vivo remarkably attenuated mechanical allodynia induced by SNL. In conclusion, peripheral nerve injury induced upregulation of OX47 in the extracellular matrix of DRG. RNA interference against OX47 significantly suppressed the expression of OX47 mRNA and the development of mechanical allodynia. The altered expression of OX47 may contribute to the development of neuropathic pain after nerve injury.

The Anti-Nociception Effect of Dezocine in a Rat Neuropathic Pain Model

The treatment of neuropathic pain (NP) currently remains clinically challenging. In an attempt to identify novel targets of known opioids, we found that dezocine, a non-addictive opioid, inhibits norepinephrine and serotonin reuptake through their transporter proteins which open the potential for dezocine to manage NP. In the present study, the effect of dezocine on NP was observed in a rat model of chronic constriction injury (CCI). The paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were used to evaluate thermal hyperalgesia and mechanic allodynia for nociceptive response. PWL and PWT tests were performed at 11:00 AM starting from 1 day before CCI surgery and 1, 3, 7, 10 days after right sciatic nerve ligation in the presence or absence of daily intraperitoneal injection of dezocine. The results demonstrated that the CCI-induced thermal and mechanical pain hypersensitivity was attenuated by dezocine significantly and persistently without sign of tolerance, indicating that dezocine could be an alternative medication for the treatment of NP. Clinical trial to confirm such discovery is warranted.

The elusive rat model of conditioned placebo analgesia

Recent research on human placebo analgesia has suggested the need for rodent models to further elucidate the neural substrates of the placebo effect. This series of 3 experiments therefore was performed in an attempt to develop a model of placebo analgesia in rats. In each study, female Sprague-Dawley rats received an L5 spinal nerve ligation to induce a neuropathic pain condition. Each rat then underwent a 4-day conditioning procedure in which an active analgesic drug or its vehicle (unconditioned stimulus) was associated with the following cues (conditioned stimuli): novel testing room (environmental), vanilla scent cue (olfactory), dim incandescent lighting (visual), restraint procedure/injection (tactile), and time of day and injection-test latency (temporal). The analgesics for each experiment were as follows: Experiment 1 used 90mg/kg gabapentin, experiment 2 used 3mg/kg loperamide hydrochloride, and experiment 3 used 6mg/kg morphine sulfate. On the following test day, half of the animals received the opposite treatment, resulting in 4 conditioning manipulations: drug/drug, drug/vehicle, vehicle/drug, and vehicle/vehicle. Nociceptive thresholds were assessed with the mechanical paw withdrawal threshold test each day after the conditioning procedure. In all 3 experiments, no significant differences were detected on test day between control and placebo groups, indicating a lack of a conditioned placebo analgesic response. Our results contrast with prior research that implies the existence of a reliable and robust response to placebo treatment. We conclude that placebo analgesia in rats is not particularly robust and that it is difficult to achieve using conventional procedures and proper experimental design.

Ketamine reduces the induced spinal p38 MAPK and pro-inflammatory cytokines in a neuropathic rats.

BackgroundNeuropathic rats created by spinal nerve ligation are known to show higher levels of p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase p44/42 (ERK 1/2) of the mitogen-activated protein kinases (MAPKs). The authors of this study aimed to understand the effect of ketamine on p38 MAPK and inflammatory responses, as well as its effect on the development of neuropathic pain.MethodsThe neuropathic rats were prepared by Chung's method with Sprague-Dawley rats. The research was carried out on three groups, a sham-operated group, a neuropathic pain and normal saline (NP + NS) group, and a neuropathic pain and ketamine (NP + Keta) group. The normal saline or ketamine was infused into the neuropathic rats through a mini-osmotic pump implanted in the subcutaneous space. After a week, the quantities of phospho-p38, p38 MAPK and pro-inflammatory cytokines were measured and compared through western blots and reverse transcriptase-polymerase chain reaction.ResultsIn comparison to the control group, the NP + NS group showed a significant increase of phospho-p38 and p38 MAPK, as well as of the proinflammatory cytokines, tumor necrosis factor α (TNFα), and intercellular adhesion molecule 1 (ICAM1). However, in the NP + Keta group, phospho-p38, p38 MAPK and TNFα and, ICAM1 were reduced in comparison to the NP + NS group. The paw withdrawal threshold test also showed the trend of recovery from the mechanical allodynia in the NP + Keta group.ConclusionsIn the development of neuropathic pain, p38 MAPK and inflammatory responses are significantly related, and the use of ketamine reduces p38 MAPK and proinflammatory cytokines. Thus, the adequate use of ketamine could be effective for the prevention and treatment of neuropathic pain following peripheral injury.

Calcitonin Gene-Related Peptide Receptor Activation Produces PKA- and PKC-Dependent Mechanical Hyperalgesia and Central Sensitization

Calcitonin gene-related peptide (CGRP), acting through CGRP receptors, produces behavioral signs of mechanical hyperalgesia in rats and sensitization of wide dynamic range (WDR) neurons in the spinal cord dorsal horn. Although involvement of CGRP receptors in central sensitization has been confirmed, the second-messenger systems activated by CGRP receptor stimulation and involved in pain transmission are not clear. This study tested whether the hyperalgesia and sensitizing effects of CGRP receptor activation on WDR neurons are mediated by protein kinase A or C (PKA or PKC) signaling. Intrathecal injection of CGRP in rats produced mechanical hyperalgesia, as shown by paw withdrawal threshold tests. CGRP-induced hyperalgesia was attenuated significantly by the CGRP1 receptor antagonist, CGRP8-37. The effect was also attenuated significantly by a PKA inhibitor (H89) or a PKC inhibitor (chelerythrine chloride). Electrophysiological experiments demonstrated that superfusion of the spinal cord with CGRP-induced sensitization of spinal dorsal horn neurons. The CGRP effect could be blocked by CGRP8-37. Either a PKA or PKC inhibitor (H89 or chelerythrine) also attenuated this effect of CGRP. These results are consistent with the hypothesis that CGRP produces hyperalgesia by a direct action on CGRP1 receptors in the spinal cord dorsal horn and suggest that the effects of CGRP are mediated by both PKA and PKC second-messenger pathways.

Central sensitization: CGRP receptor activation produces PKA and PKC dependent mechanical hyperalgesia and central sensitization

Calcitonin gene-related peptide (CGRP), acting through CGRP1 receptors, produced behavioral signs of hyperalgesia in rats and sensitization of wide dynamic range (WDR) neuron in the spinal cord dorsal horn (Sun, et al., 2003). Although, involvement of CGRP receptors in central sensitization has been confirmed, the second messenger systems activated by CGRP receptor stimulation and that are involved in pain transmission are not clear. This study tested whether the hyperalgesic and wide dynamic range (WDR) neuron sensitizing effects of CGRP receptor activation is medicated by PKA or PKC signaling. Intrathecal injection of CGRP in rats produced mechanical hyperalgesia, as shown by paw withdrawal threshold tests. CGRP-induced hyperalgesia was attenuated significantly by the CGRP1 receptor antagonist, CGRP8-37. The effect was attenuated significantly by a protein kinase A (PKA) inhibitor (H89) or a protein kinase C (PKC) inhibitor (chelerythrine chloride). Electrophysiological experiments demonstrated that superfusion of the spinal cord with CGRP induced the sensitization of spinal dorsal horn neurons. The effect could be blocked by CGRP8-37. Either a PKA or a PKC inhibitor (H89 or chelerythrine) also attenuated this effect of CGRP. These results are consistent with the hypothesis that CGRP produces hyperalgesia by a direct action on CGRP receptors in the spinal cord dorsal horn and that this effect of CGRP is dependent on PKA and PKC signaling. In conclusion, CGRP produces mechanical hyperalgesia and sensitizes WDR neurons in dorsal horn in the absence of nerve injury via an action at CGRP1 receptors. These effects of CGRP are mediated by both PKA and PKC second-messenger pathways. Supported by NIH grants NS09743 and NS11255.