Paw Withdrawal Frequency Research Articles

Histopathological Changes in a Rat Model of Diabetic Neuropathy

This study was done to investigate the histopathological Changes in a Rat Model of Diabetic Neuropathy. The 24 male rats used in this investigation were split into two groups of 12 animals. The first group (G1), which consisted of healthy individuals, was given standard saline. Normal saline was administered after G2 (STZ group) received STZ at a dosage of 60 mg/kg. After rats were starved overnight, a single intraperitoneal injection of 60 mg/kg of freshly produced STZ was given to induce diabetes. (0.1 M, pH 4.5) Sodium Citrate Buffer was used to dissolve STZ. On the third day after the injection of STZ, a digital glucometer was used to measure the blood glucose levels of 12-hour fasting rats; hyperglycemia was defined as values greater than 250 mg/dl. In order to assess neuropathic pain, cold allodynia was calculated by counting the number of times the foot withdrew in response to cold stimuli applied to the back of the hand. The sciatic nerve was carefully removed after the sacrifice (after 2 months) and preserved in 10% formalin for histopathological study. Normal blood glucose levels were maintained in the control rats throughout the study. However, G2 exhibited much greater FBG levels than healthy animals. In the 2-month study, the G2 maintained FBG levels above 250 mg/dl. The score for severity of STZ on sciatic nerves showed a significant difference between treated group as compared with G1. No paw reactions were seen when intact animals were subjected to plantar surface acetone. The experimental groups did not show any notable reactions to acetone on the tenth day after the diagnosis of diabetes. On days 20, 30, 40, 50 and 60 after diabetes confirmation, there were significant differences in paw withdrawal frequency between the G1 and G2 (p<0.001). Within the control group, nerve fibres were found to be regularly arranged and there was no evidence of axonal swelling. However, in the G2 group, nerve fibres were found to be significantly disorganised, and there was a notable increase in the number of Schwann cells in the sciatic nerves, suggesting damage, in comparison to the control group. In conclusion, potentially via antinociceptive, antihyperglycemic, antioxidant, and neuro-regeneration boosting pathways in STZ-induced diabetic peripheral disease.

Electroacupuncture promotes macrophage/microglial M2 polarization and suppresses inflammatory pain through AMPK.

Inflammatory pain, the most prevalent disease globally, remains challenging to manage. Electroacupuncture emerges as an effective therapy, yet its underlying mechanisms are not fully understood. This study investigates whether adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-regulated silent information regulator 1 (SIRT1) contributes to electroacupuncture's antinociceptive effects by modulating macrophage/microglial polarization in the spinal dorsal horn of a mouse model of inflammatory pain. In this study, mice, introduced to inflammatory pain through subcutaneous injections of complete freund's adjuvant (CFA) in the plantar area, underwent electroacupuncture therapy every alternate day for 30-min sessions. The assessment of mechanical allodynia and thermal hyperalgesia in these subjects was carried out using paw withdrawal frequency and paw withdrawal latency measurements, respectively. Western blot analysis measured levels of AMPK, phosphorylation-adenosine 5'-monophosphate (AMP)-activated protein kinase, SIRT1, inducible nitric oxide synthase, cluster of differentiation 86, arginase 1, and interleukin 10. In contrast to the group treated solely with CFA, the cohort receiving both CFA and electroacupuncture demonstrated notable decreases in both thermal hyperalgesia and mechanical allodynia. This was accompanied by a marked enhancement in AMPK phosphorylation levels. AMPK knockdown reversed electroacupuncture's analgesic effects and reduced M2 macrophage/microglial polarization enhancement. Additionally, AMPK knockdown significantly weakened electroacupuncture-induced SIRT1 upregulation, and EX-527 injection attenuated electroacupuncture's facilitation of M2 macrophage/microglial polarization without affecting AMPK phosphorylation levels. Furthermore, combining electroacupuncture with SRT1720 enhanced the analgesic effect of SRT1720. Our findings suggest that AMPK regulation of SIRT1 plays a critical role in electroacupuncture's antinociceptive effect through the promotion of M2 macrophage/microglial polarization.

Classical cannabinoid receptors as target in cancer-induced bone pain: a systematic review, meta-analysis and bioinformatics validation

To test the hypothesis that genetic and pharmacological modulation of the classical cannabinoid type 1 (CB1) and 2 (CB2) receptors attenuate cancer-induced bone pain, we searched Medline, Web of Science and Scopus for relevant skeletal and non-skeletal cancer studies from inception to July 28, 2022. We identified 29 animal and 35 human studies. In mice, a meta-analysis of pooled studies showed that treatment of osteolysis-bearing males with the endocannabinoids AEA and 2-AG (mean difference [MD] − 24.83, 95% confidence interval [95%CI] − 34.89, − 14.76, p < 0.00001) or the synthetic cannabinoid (CB) agonists ACPA, WIN55,212-2, CP55,940 (CB1/2-non-selective) and AM1241 (CB2-selective) (MD − 28.73, 95%CI − 45.43, − 12.02, p = 0.0008) are associated with significant reduction in paw withdrawal frequency. Consistently, the synthetic agonists AM1241 and JWH015 (CB2-selective) increased paw withdrawal threshold (MD 0.89, 95%CI 0.79, 0.99, p < 0.00001), and ACEA (CB1-selective), AM1241 and JWH015 (CB2-selective) reduced spontaneous flinches (MD − 4.85, 95%CI − 6.74, − 2.96, p < 0. 00001) in osteolysis-bearing male mice. In rats, significant increase in paw withdrawal threshold is associated with the administration of ACEA and WIN55,212-2 (CB1/2-non-selective), JWH015 and AM1241 (CB2-selective) in osteolysis-bearing females (MD 8.18, 95%CI 6.14, 10.21, p < 0.00001), and treatment with AM1241 (CB2-selective) increased paw withdrawal thermal latency in males (mean difference [MD]: 3.94, 95%CI 2.13, 5.75, p < 0.0001), confirming the analgesic capabilities of CB1/2 ligands in rodents. In human, treatment of cancer patients with medical cannabis (standardized MD − 0.19, 95%CI − 0.35, − 0.02, p = 0.03) and the plant-derived delta-9-THC (20 mg) (MD 3.29, CI 2.24, 4.33, p < 0.00001) or its synthetic derivative NIB (4 mg) (MD 2.55, 95%CI 1.58, 3.51, p < 0.00001) are associated with reduction in pain intensity. Bioinformatics validation of KEGG, GO and MPO pathway, function and process enrichment analysis of mouse, rat and human data revealed that CB1 and CB2 receptors are enriched in a cocktail of nociceptive and sensory perception, inflammatory, immune-modulatory, and cancer pathways. Thus, we cautiously conclude that pharmacological modulators of CB1/2 receptors show promise in the treatment of cancer-induced bone pain, however further assessment of their effects on bone pain in genetically engineered animal models and cancer patients is warranted.

Silencing of secreted phosphoprotein 1 attenuates sciatic nerve injury-induced neuropathic pain: Regulating extracellular signal-regulated kinase and neuroinflammatory signaling pathways.

Neuropathic pain (NP) is a chronic pathological pain that affects the quality of life and is a huge medical burden for affected patients. In this study, we aimed to explore the effects of secreted phosphoprotein 1 (SPP1) on NP. We established a chronic constriction injury (CCI) rat model, knocked down SPP1 via an intrathecal injection, and/or activated the extracellular signal-regulated kinase (ERK) pathway with insulin-like growth factor 1 (IGF-1) treatment. Pain behaviors, including paw withdrawal threshold (PWT), paw withdrawal latency (PWL), lifting number, and frequency, were assessed. After sacrificing rats, the L4-L5 dorsal root ganglion was collected. Then, SPP1 levels were determined using quantitative polymerase chain reaction (qPCR) and western blot analysis. The levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-β were determined using qPCR and enzyme-linked immunosorbent assay. The levels of ERK pathway factors were determined via western blot analysis. We found that CCI decreased PWT and PWL, increased the lifting number and frequency, and upregulated SPP1 levels. The loss of SPP1 reversed these CCI-induced effects. Additionally, CCI upregulated IL-1β, TNF-α, IL-6, EGF, and VEGF levels, downregulated TGF-β levels, and activated the ERK pathway, while silencing of SPP1 abrogated these CCI-induced effects. Moreover, IGF-1 treatment reversed the effects of SPP1 loss. The data indicate that silencing SPP1 attenuates NP via inactivation of the ERK pathway, suggesting that SPP1 may be a promising target for NP treatment.

Obacunone Alleviates Inflammatory Pain by Promoting M2 Microglial Polarization and by Activating Nrf2/HO-1 Signaling Pathway.

Treating inflammatory pain (IP) continues to pose clinical challenge, because of the lack of effective pharmacological interventions. Microglial polarization serves as pivotal determinant in IP progress. Obacunone (OB), a low-molecular-weight compound with a diverse array of biological functions, having reported as an activator of nuclear factor E2-related factor 2 (Nrf2), exhibits anti-inflammatory property. However, it remains uncertain whether OB can alleviate IP by facilitating the transition of microglial polarization from the M1 to M2 state through modulating Nrf2/ heme oxygenase-1 (HO-1) pathway. We induced an mice IP model by subcutaneously administering Complete Freund's Adjuvant (CFA) into the hind paw. Paw withdrawal latency (PWL) in seconds (s) and paw withdrawal frequency (PWF) were employed to evaluate the establishment of the IP model, while a caliper was used to measure the maximal dorsoventral thickness of the mice paw. Nerve injury was assessed by Hematoxylin-Eosin (HE) Staining. Western blot and got conducted for detection of M1/M2 microglial polarization markers, Nrf2 and HO-1 in spinal cord tissues respectively. In comparison to the control cohort, PWF, M1 phenotype marker iNOS, CD86, paw thickness increased significantly within CFA cohort, while PWL, M2 phenotype marker Arg-1, interleukin-10 (IL-10) decreased in the CFA group. In comparison to model cohort, OB treatment decreased PWF, paw thickness, M1 phenotype marker iNOS, CD86 significantly, while PWL, M2 phenotype marker Arg-1, IL-10, Nrf2, HO-1 increased significantly. The morphological injuries of sciatic nerve in CFA mice were obviously improved by OB treatment. OB inhibited the release of M1-related IL-1β, CXCL1 but promoted M2-related TGF-β, IL-10 in serum in CFA mice. The intervention of the Nrf2 inhibitor ML385 mitigated analgesic effect of OB. We demonstrate that OB is able to attenuate inflammatory pain via promoting microglia polarization from M1 to M2 and enhancing Nrf2/HO-1 signal. OB treatment may be a potential alternative agent in the treatment of IP.

Translational PK-PD/TD modeling of antitumor effects and peripheral neuropathy in gemcitabine and nab-paclitaxel chemotherapy from xenograft mice to patients for optimal dose and schedule.

Gemcitabine and nab-paclitaxel (GnP) treatment, the standard first-line chemotherapy for unresectable pancreatic cancer, often causes peripheral neuropathy (PN). To develop alternative dosing strategies to avoid severe PN, understanding the relationship between pharmacokinetics (PK) and pharmacodynamics/toxicodynamics (PD/TD) is necessary. We established a PK-PD/TD model of GnP treatment to develop an optimal dose schedule. A mouse xenograft model of human pancreatic cancer was generated to measure drug concentrations in the plasma and tumor, antitumor effects, and PN after GnP treatment. The Simeoni tumor growth inhibition model with tumor concentrations and empirical indirect response models were used for the PD and TD models, respectively. Clinical outcomes were predicted with reported population estimates of PK parameters in cancer patients. The PK-PD/TD model simultaneously described the observed tumor volume and paw withdrawal frequency in the von Frey test. For the standard GnP regimen, the model predicted clinical overall response (75.1%), which was overestimated compared to that in a recent phase II study (42.1%) but lower than the observed disease control rate (96.5%). Model simulation showed that dose reduction to less than 40% GnP dose was not effective; a change of dose schedule from every week for 3weeks to every 2weeks was a more favorable approach than dose reduction to 60% every week. The PK-PD/TD model-based translational approach provides a guide for optimal dose determination to avoid severe PN while maintaining antitumor effects during GnP chemotherapy. Further research is needed to enhance its applicability and potential for combination chemotherapy regimens.

Comfort Therapy with Companionship Improves Learning Memory and Reduces Hyperalgesia in Sickle Mice

Chronic and unpredictable acute pain coexist with cognitive dysfunction in sickle cell disease (SCD). Managing chronic pain is crucial, though efforts are complicated by both physiological and affective contributors to pain. The affective state, which is severely impacted by the environment including stigma and social isolation, plays a substantial role in pain perception and may further exacerbate psychological factors. Therefore, we examined if companionship and social isolation influence cognitive function and pain in SCD. In the present study, we examined the effects of social isolation on hyperalgesia, learning memory, and anxiety in humanized transgenic homozygous BERK mouse model of SCD. At ~3.5 months of age, BERK male mice were paired with age-matched female BERK companions (1 male + 1 female/cage; called companion group) and another group of BERK males were placed into isolation (one male/cage; called isolation group) for ~14 months. Isolation and companion groups were maintained in general housing conditions in the same room. Both groups were assessed for body weight, hyperalgesia and cognitive behaviors. Mechanical hyperalgesia was assessed by counting paw withdrawal frequency (PWF) following 10 applications of a 1.0 g von Frey monofilament (Semmes Weinstein, Stoelting, Wood Dale, IL, USA) to the plantar surface of the hind paw. Cold hyperalgesia was assessed following placement of mice onto a 4°C cold plate chamber (Stoelting) and by measuring PWF over a 2-minute period. Musculoskeletal hyperalgesia was measured by testing grip force of each mouse's peak forepaw grip to a computerized grip force meter (SA Maier, Milwaukee, WI, USA) and normalizing by body weight. The cognitive behavioral assessment consisted of an open field test to assess anxiety-related behaviors, which measured time spent and number of crosses through a central region. Working memory was assessed using novel object recognition and novel placement recognition tests to measure discrimination of novel objects and location. Data are shown as mean ± SEM and comparisons between companionship and isolation groups were performed using unpaired, two-tailed T-tests to determine statistical significance (P&amp;lt;0.05) We observed a significant increase in the body weights of males housed with females ( Companion group) compared to males in isolation (p&amp;lt;0.05). It is likely that males living with a companion were eating more compared to those under isolation. Isolated male sickle mice experience greater hyperalgesia than male mice housed with a female companion. Isolation induces greater PWF to mechanical (P&amp;lt;0.05) and cold (P&amp;lt;0.005) stimuli, indicating exacerbated hypersensitivity to mechanical and cold stimuli. Spontaneous, non-evoked musculoskeletal hyperalgesia was not significantly altered by isolation (P&amp;gt;0.05). Isolation also appeared to impact working and learning memory. Novel object recognition and novel object placement tests revealed reduced discrimination ratios in isolated mice (~25%, P&amp;lt;0.05 and ~25%, P&amp;lt;0.05), which are suggestive of impaired learning and memory function compared to mice with companionship. Open field test showed no significant difference (P&amp;gt;0.05) between isolation and companionship groups in time spent and number of crosses in the open field center area, indicating no detectable differences in anxiety-like behaviors. According to cognitive tests, mice in companionship showed greater novel object and place detection and greater cognitive ability compared to those in isolation. For the first time we show the causal role of social isolation on hyperalgesia and memory in a humanized mouse model of SCD. “ Comfort therapy” with companionship can ameliorate the perception of pain through the higher brain centres. It is also likely that mice in companionship eat more as evinced by a significant increase in their weight, which may provide additional comfort and meet the increased nutritional/calorie demands of SCD due to altered metabolism. Our observations suggest that “ comfort therapy” with improved living conditions and reducing isolation and loneliness may improve pain-related outcomes in SCD.

Mechanism-Based Targeting of Sickle Cell Pathobiology and Pain with Novel Transdermal Curcumin

Pain is a debilitating consequence of sickle cell disease (SCD). We examined the potential of novel topical transdermal curcumin (TDC) gel with transdermal systemic delivery to target pain and SCD pathobiology. Curcumin is an antioxidant and anti-inflammatory polyphenol with therapeutic potential but has poor oral bioavailability. TDC gel (Vasceptor TM, Vascarta Inc.), containing 0.1 M of curcuminoids (derived from Curcugen, Dolcas-Biotech, LLC), or vehicle were applied (0.1 mL) topically by gentle rubbing to the abdomen on alternating days for 3 weeks followed by analysis of blood, organ pathology and skin secretome. Using high-performance liquid chromatography, we found that curcumin levels peaked in the plasma (7.87 µg/mL) and blood cells (6.78 µg/mL) 60 min following administration in C57BL/6 mice, thus, showing efficient bioavailability. Next, we used humanized ‘ sickle‘, homozygous transgenic mice ( HbSS-BERK) expressing &amp;gt;99% human sickle hemoglobin (HbSS) without mouse α- and β-globins. These mice mimic the pathobiology and features of pain observed in persons with SCD. We observed that TDC application in male and female HbSS led to a significant decrease in hyperalgesia over the 21 days (D) of treatment. Mechanical hyperalgesia significantly decreased after 14D of treatment in males and after 21D in females (p&amp;lt;.05 and p&amp;lt;.01, respectively Vs. BL). Similarly, cold hyperalgesia (paw withdrawal frequency on cold plate at 4˚C) decreased significantly in males after 24 hours and in females after 7D of TDC treatment (p&amp;lt;.05 and .01, respectively Vs. BL). Non-evoked cold avoidance showed that males spent significantly more time in the colder chamber at 23ºC on 14D, and females did so on 21D after TDC treatment (p&amp;lt;.001 &amp; p&amp;lt;.01, respectively Vs. BL). Throughout 21D TDC treatment, females showed significantly lower analgesic response in cold hyperalgesia compared to male HbSS, similar to relatively poor response to analgesia in female Vs. male individuals with SCD. The amelioration of hyperalgesia for 21D with TDC suggests a disease-modifying effect. Indeed, we observed a significant increase in hematocrit (p&amp;lt;.05), a reduction in reticulocytes (p&amp;lt;.05), and an appreciable increase (43.2%) in Hb and decreased lactate dehydrogenase activity in the blood of TDC-treated HbSS mice Vs. vehicle (p&amp;lt;.01). RBCs from TDC treated HbSS mice showed enhanced ATP levels (p &amp;lt;.01) and reduced protein carbonylation, both biomarkers of protein oxidation (p&amp;lt;.045) Vs RBCs from vehicle-treated HbSS. Histopathology showed a significant reduction in vascular congestion and iron deposits in TDC-treated male HbSS compared to vehicle in the spleen (p&amp;lt;.0003 &amp; p&amp;lt;.001, respectively) and in % fields involved in hepatic infarcts and iron deposits (p&amp;lt;.02 &amp; p&amp;lt;.044, respectively). Together, decreased hemolysis, increased Hb, hematocrit, and ATP suggest RBC stabilization, improved mitochondrial metabolism, and reduced oxidative stress and organ damage, leading to a disease-modifying effect of TDC. TDC also significantly reduced global inflammatory marker serum amyloid-P (p&amp;lt;.05) and inflammatory cytokines in the skin secretome with a concomitant reduction in interleukins [2 (p&amp;lt;.05), 4 (p&amp;lt;.01), and 6 (p&amp;lt;.01)], monocyte chemoattractant protein 1 (MCP-1; p&amp;lt;.01), interferon-gamma (IFN-γ; p&amp;lt;.05), granulocyte macrophage-colony stimulating factor (GM-CSF; p&amp;lt;.01), and regulated on activation, normal T-cell expressed and secreted protein (RANTES; p&amp;lt;.05) compared to vehicle. MCP-1 contributes to neuropathic pain and inflammation; GM-CSF stimulates granulocyte differentiation and growth. Both RANTES and MCP-1 may also activate mast cells. TDC led to a significant decrease in cutaneous mast cell degranulation (p&amp;lt;.001) Vs. vehicle in sickle mice. These data strongly suggest an anti-inflammatory effect of TDCby targeting the release of cytokines and inhibiting granulocyte activity. TDC significantly ameliorates hyperalgesia (chronic pain), inflammation, hemolysis, oxidative stress, and organ damage and improves mitochondrial function and hematological parameters of SCD pathobiology, indicative of disease modifying and anti-nociceptive effects. The results suggest if started early in life, the use of an easy-to-deploy novel TDC formulation, with increased bioavailability and non-toxic properties that directly target sickle cell pathobiology, may prevent chronic pain in SCD.

Palmitoylethanolamide Modulates Gait By Targeting Purkinje Cells in the Brain of Sickle Mice

Chronic pain is a major comorbidity of sickle cell disease (SCD). Transgenic humanized (BERK) sickle mice recapitulate many clinical features of SCD, including chronic pain. Motor impairments concomitant with increased pyknotic cerebellar Purkinje cells occur in BERK sickle mice. Neurological, neuropathic, and immunological factors, including Purkinje cell damage and chronic inflammation, may contribute to peripheral and central mechanisms that drive gait dysfunction. Pathological changes in Purkinje cells of sickle mice include a significantly greater number of pyknotic cells with smudged nuclei, condensed chromatin, and lack of well-formed nucleoli, &amp; may contribute to alterations in gait as Purkinje cells regulate sensory-motor functions. Subjects with SCD often use cannabinoids for pain relief, but the intoxicating effects and social stigma associated with their use represent barriers to utilizing this treatment. Palmitoylethanolamide (PEA) is a non-intoxicating endocannabinoid-like lipid mediator naturally produced throughout the body that may act via indirect cannabinoid receptor activation. In clinical studies associated with movement and pain, including chronic arthritic and multiple sclerosis, PEA has demonstrated a reduction of motor dysfunction and pain. Therefore, we examined the effect of PEA on hyperalgesia and gait in humanized HbSS-BERK sickle (HbSS) mice. We utilized ~6-month-old female, HbSS mice, expressing &amp;gt;99% human α- and ßS- sickle hemoglobin and complete knockout of murine α- and β-globins. Mice were treated with PEA (ip, 20 mg/kg/d) or vehicle (7.5% DMSO, 7.5% Tween 20, in sterile saline) for 2 weeks (2-wks). Gait measurements were gathered for each mouse at baseline (BL) and 2-wks post treatment during normal walking in MouseWalker's transparent corridor with a floor panel mounted with LED lights to produce a detectable touch sensor. Physical contact of paw pads with the surface during natural walking disrupts the light path, causing frustrated total internal reflection detected by a high-speed camera and analyzed using artificial intelligence-based algorithms for gait parameters. Mechanical hyperalgesia was analyzed as paw withdrawal frequency (PWF) in response to 10 applications of 1.0 g von Frey monofilaments. Complete blood counts and brain histopathology on hematoxylin- and eosin-stained sections were evaluated after 2-wks post-treatment. We observed that PEA ameliorates features of dysfunctional gait in HbSS mice. PEA-treated mice show increased limb swing speed (~30%, P&amp;lt;0.01) and shortened swing duration (20%, P&amp;lt;0.05) without affecting walking speed (P &amp;gt;0.05) Vs. vehicle-treated mice. Also, PEA significantly reduced body instability (~75%, P&amp;lt;0.05) and improved stance duration (~65%, P&amp;lt;0.05) Vs. BL. This suggests improved limb coordination and reduced sensitivity to mechanical forces during walking. Complementary to gait changes, PEA significantly decreased mechanical hyperalgesia, indicated by lower PWF, 1-hour following initial treatment and following 2-wks post-treatment (~55%, P&amp;lt; 0.001 and ~65%, P&amp;lt; 0.001, respectively) Vs. BL. PEA decreased the number of circulating white blood cells (P&amp;lt; 0.05), with the greatest reduction in circulating lymphocytes (P&amp;lt; 0.1). PEA had no adverse effects on organ weight Vs. vehicle treatment. PEA-treated mice showed a significant reduction in pyknotic cerebellar Purkinje cells (~23.9% p&amp;lt;.006) in the brain Vs. vehicle. Thus, Purkinje cell pathology may contribute to impaired gait, and PEA may improve motor coordination by reducing Purkinje cell damage. We provide the first evidence that PEA administration in a humanized sickle mouse model improves features of gait, restores Purkinje cell morphology, and reduces inflammation. Our data demonstrate that PEA may have a disease-modifying effect and restoration of sensory-motor function in SCD. Impairment in gait may also be due to avascular necrosis. Therefore gait measurements may provide a non-invasive analysis of movement associated pathology and pain as well as effectiveness of targeted interventions.

P66shc in the spinal cord is an important contributor in complete Freund's adjuvant induced inflammatory pain in mice

PurposeThe aim of this study is to investigate whether p66shc is involved in inflammatory pain and the potential molecular mechanisms of p66shc in inflammatory pain. MethodsInflammatory pain model was established by complete Freund's adjuvant (CFA) injection. Paw withdrawal latency (PWL) and paw withdrawal frequency (PWF) was recorded. The expression of spinal p66shc were determined by immunohistochemical staining, immunofluorescence staining. P66shc knockdown was performed by an adeno-associated virus (AAV) vector infusion. NLRP3 inflammasome complexes were determined by Western blot. DHE staining was used to evaluate reactive oxygen species (ROS) generation. ResultsP66Shc expression was progressively elevated in spinal cord of inflammatory pain mice, and p66Shc knockdown in vivo significantly attenuated CFA injection triggers hyperalgesia. Furthermore, knockdown of p66Shc significantly inhibited ROS production and NOD-like receptor protein 3 (NLRP3) inflammasome activation, which were reversed by a ROS donor (t-BOOH). However, post-treatment with nigericin, a agonist of NLRP3, reversed AAV-shP66shc analgesic effect. ConclusionSpinal p66shc may facilitate the development of inflammatory pain by promoting the activation of NLRP3 inflammasome through ROS.

Colchicine Reduces Inflammation in a Humanized Mouse Model of Sickle Cell Disease

Introduction Inflammation, marked by a "cytokine storm" of circulating proinflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor- α (TNF-α), and C-reactive protein (CRP), and increases in intracellular NLRP3 inflammasomes is an important component of sickle cell disease (SCD). Although current treatments have limited anti-inflammatory effects, targeting inflammation is a promising but unrealized treatment approach. Colchicine, a tubulin inhibitor, is a promising anti-inflammatory agent for SCD. It is useful in gout, familial Mediterranean fever, pericarditis and other inflammatory diseases. In these contexts, it often reduces the same inflammatory proteins and cellular components that are elevated in SCD. We sought preliminary evidence for colchicine as an anti-inflammatory treatment for SCD. Methods Humanized sickle mice (HbSS-BERK) are knockout for murine α and β globins and express human α and βS globins (>99% βS) on a mixed genetic background. HbSS-BERK have severe disease including hyperalgesia. Experiments were performed following protocols approved by the Institutional Animal Care and Use Committee. Female mice of ~ 3.5 months of age received either colchicine 100 µg/kg body weight or vehicle (phosphate-buffered saline) intraperitoneally daily for 14 d. Pain-related behavior testing was performed before treatment and 1 h, 24 h, 7 d, and 14 d after the 1st dose of study agent. Mechanical hyperalgesia was tested by paw withdrawal frequency (PWF) in response to the application of a von Frey monofilament to the mid-plantar surface of the hind paw. Musculoskeletal/deep tissue hyperalgesia was tested by measurement of the peak tensile force exerted by a forelimb upon traction. Cold hyperalgesia was tested by PWF/2 min upon placement of mice on a cold plate maintained at ~4°C. After the 14 d of dosing, mice were euthanized; blood was collected; mice were necropsied; and 4 mm diameter punch biopsies of dorsal skin were harvested and incubated in culture medium for 24 hours to allow for release of proteins into the skin secretagogue Plasma serum amyloid-P (SAP) and IL-6 were analyzed with enzyme-linked immunosorbent assays. Limited blood volumes prevented assay of other circulating proteins. Automated and manual cytometry were used to characterize circulating blood cells. Skin secretagogue was assayed for cytokines IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, monocyte chemoattractant protein 1 (MCP-1), interferon gamma (IFN-γ), TNF-α, macrophage inflammatory protein-1 alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and regulated on activation, normal T-cell expressed and secreted protein (RANTES) using Q-PlexTM microarray. Data was analyzed using conventional descriptive statistics. Results As per usual laboratory practice, we considered mouse circulating SAP levels as surrogate for human CRP levels. Following 14 d of dosing, plasma IL-6 levels were ~67% lower (~130 v ~48 µg/g protein; p<0.01), and plasma SAP levels were ~45% lower (~33 v ~18 µg/g protein; p<0.01) in colchicine-treated mice. There were no significant differences in blood cell parameters. Concentrations of IL-3, IL-1, IFN-γ, and GM-CSF were 40% (p<0.05), 20% (p<0.05), 40% (p<0.01), and 40% (p<0.01) lower, respectively, in the skin secretagogue of colchicine-treated mice. Given the chronic nature of inflammation in SCD, we did not anticipate phenotypic changes after only a 14-day exposure to colchicine. Indeed, no significant differences in hyperalgesia or mouse/organ weight were observed. We think that next steps should be dose-finding studies of colchicine in adults living with SCD followed by randomized, placebo-controlled trials with clinically relevant endpoints such as repeated measures of pain interference using a validated instrument. The anticipated lack of colchicine effect upon blood cell parameters suggests that patients would benefit more from a combination of a disease-modifying agent such as hydroxyurea in combination with colchicine.

Inhibition of angiotensin converting enzyme induces mechanical allodynia through increasing substance P expression in mice

Although emerging evidence shows that angiotensin converting enzyme (ACE) is associated with pain, it is not clear whether inhibition of ACE could affect to nociceptive transmission and which mediators are involved in this process. Here we investigated whether administration of the ACE inhibitors, captopril and enalapril increases the expression of substance P (SP) and whether this increase contributes to the induction of mechanical allodynia in mice. ACE was expressed in the lumbar dorsal root ganglion (DRG) and the superficial dorsal horn (SDH) region of the spinal cord in mice. Either intraperitoneal or intrathecal administration of the ACE inhibitors, captopril and enalapril for 10 days significantly increased the paw withdrawal frequency to innocuous mechanical stimuli and the levels of SP in both the lumbar DRG and the SDH region of the spinal cord dorsal horn. In addition, intraperitoneal administration of the SP receptor (neurokinin-1 receptor) antagonist, L-733,060 suppressed mechanical allodynia that was induced by pretreatment of captopril and enalapril. Intraplantar administration of SP for 3 days induces mechanical allodynia, and this effect was reduced by exogenous ACE administration. These findings demonstrate that inhibition of ACE increases the levels of SP in both the lumbar DRG and spinal cord dorsal horn, ultimately contributing to the induction of mechanical allodynia in mice.

The role of spinal inhibitory neuroreceptors in the antihyperalgesic effect of warm water immersion therapy

ObjectiveWarm water immersion therapy (WWIT) has been widely used in the treatment of various clinical conditions, with analgesic and anti-inflammatory effects. However, its mechanism of action has not been fully investigated. The present study analyzed the role of spinal inhibitory neuroreceptors in the antihyperalgesic effect of WWIT in an experimental model of inflammatory pain. MethodsMice were injected with complete Freund’s adjuvant (CFA; intraplantar [i.pl.]). Paw withdrawal frequency to mechanical stimuli (von Frey test) was used to determine: (1) the effect of intrathecal (i.t.) preadministration of naloxone (a non-selective opioid receptor antagonist; 5 µg/5 µl), (2); AM281 (a selective cannabinoid receptor type 1 [CB1] antagonist; 2 µg/5 µl), (3); and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; a selective adenosine A1 receptor antagonist; 10 nmol/5 µl), on the antihyperalgesic (pain-relieving) effect of WWIT against CFA-induced hyperalgesia. ResultsIntrathecal naloxone, AM281, and DPCPX significantly prevented the antihyperalgesic effect of WWIT. This study suggests the involvement of spinal (central) receptors in the antihyperalgesic effect of WWIT in a model of inflammatory pain. ConclusionsTaken together, these results suggest that opioid, CB1, and A1 spinal receptors might contribute to the pain-relieving effect of WWIT.

Exendin-4 attenuates pain-induced cognitive impairment by alleviating hippocampal neuroinflammation in a rat model of spinal nerve ligation.

Glucagon-like peptide-1 receptor has anti-apoptotic, anti-inflammatory, and neuroprotective effects. It is now recognized that the occurrence and development of chronic pain are strongly associated with anti-inflammatory responses; however, it is not clear whether glucagon-like peptide-1 receptor regulates chronic pain via anti-inflammatory mechanisms. We explored the effects of glucagon-like peptide-1 receptor on nociception, cognition, and neuroinflammation in chronic pain. A rat model of chronic pain was established using left L5 spinal nerve ligation. The glucagon-like peptide-1 receptor agonist exendin-4 was intrathecally injected into rats from 10 to 21 days after spinal nerve ligation. Electrophysiological examinations showed that, after treatment with exendin-4, paw withdrawal frequency of the left limb was significantly reduced, and pain was relieved. In addition, in the Morris water maze test, escape latency increased and the time to reach the platform decreased following exendin-4 treatment. Immunohistochemical staining and western blot assays revealed an increase in the numbers of activated microglia and astrocytes in the dentate gyrus of rat hippocampus, as well as an increase in the expression of tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6. All of these effects could be reversed by exendin-4 treatment. These findings suggest that exendin-4 can alleviate pain-induced neuroinflammatory responses and promote the recovery of cognitive function via the glucagon-like peptide-1 receptor pathway. All experimental procedures and protocols were approved by the Experimental Animal Ethics Committee of Renmin Hospital of Wuhan University of China (approval No. WDRM 20171214) on September 22, 2017.

Role of ZHX2 in regulating dorsal root ganglion μ-opioid receptor expression in mice with peripheral nerve injuryinduced pain hypersensitivity

To investigate the role of zinc-fingers and homeoboxes 2 (ZHX2) in regulating μ-opioid receptor expression in the dorsal root ganglion (DRG) in mice with peripheral nerve injury-induced pain hypersensitivity. Forty-eight male adult C57BL6J mice were randomized into 4 groups and subjected to chronic constriction injury (CCI) of the sciatic nerve or sham operation followed by microinjection of a specific small interfering RNA (siRNA) of ZHX2 or a negative control siRNA sequence (siNC) into the DRG. Seven days later, the mice were examined for changes in the hind paw withdrawal frequency (PWF), after which the DRG tissue was collected for detecting the expressions of μ-opioid receptor at the mRNA and protein levels using RT-qPCR and Western blotting. In another experiment, the DRG tissues were collected from 6 mice (21-day-old) for primary culture of the DRG neurons, which were transfected with ZHX2 siRNA or the siNC to observe the changes in the expressions of ZHX2 and μ-pioid receptor. Microinjection of ZHX2 siRNA into the ipsilateral L3 and L4 DRGs significantly reversed CCI-induced μ-pioid receptor downregulation in the injured DRG and alleviated CCI-induced mechanical allodynia in the mice. In the cell experiment, ZHX2 knockdown obviously upregulated the mRNA and protein expressions of opioid receptor in the primary cultured DRG neurons. ZHX2 knockdown in the DRG reverses CCI-induced down-regulation of μ opioid receptor to alleviate periphery nerve injury-induced pain hypersensitivity in mice.

Dermorphin [D-Arg2, Lys4] (1-4) amide inhibits below-level heat hypersensitivity in mice after contusive thoracic spinal cord injury.

Opioid use for chronic pain is limited by severe central adverse effects. We examined whether activating mu-opioid receptors (MORs) in the peripheral nervous system attenuates spinal cord injury (SCI) pain-like behavior in mice. We produced a contusive SCI at the T10 vertebral level and examined motor and sensory dysfunction for 6 weeks. At 6 weeks, we tested the effect of subcutaneous (s.c.) injection of dermorphin [D-Arg2, Lys4] (1-4) amide (DALDA), a peripherally acting MOR-preferring agonist, on mechanical and heat hypersensitivity. Basso mouse scale score was significantly decreased after SCI, and mice showed hypersensitivity to mechanical and heat stimulation at the hind paw beginning at 2 weeks, as indicated by increased paw withdrawal frequency to mechanical stimulation and decreased paw withdrawal latency to heat stimulation. In wild-type SCI mice, DALDA (1 mg/kg, s.c.) attenuated heat but not mechanical hypersensitivity. The effect was blocked by pretreatment with an intraperitoneal injection of methylnaltrexone (5 mg/kg), a peripherally restricted opioid receptor antagonist, and was also diminished in Pirt-MOR conditional knockout mice. DALDA did not adversely affect exploratory activity or induced preference to drug treatment in SCI mice. In vivo calcium imaging showed that DALDA (1, 10 mg/kg, s.c.) inhibited responses of small dorsal root ganglion neurons to noxious heat stimulation in Pirt-GCaMP6s mice after SCI. Western blot analysis showed upregulation of MOR in the lumbar spinal cord and sciatic nerves at 6 weeks after SCI. Our findings suggest that peripherally acting MOR agonist may inhibit heat hypersensitivity below the injury level with minimal adverse effects.

Microinjection of histamine and its H3 receptor agonist and antagonist into the agranular insular cortex influence sensory and affective components of neuropathic pain in rats

Many areas of the brain along with neurotransmitters involve in processing of nociceptive, emotional and cognitive dimensions of neuropathic pain. Brian neuronal histamine through H1, H2, H3 and H4 receptors mediates many physiological functions such as cognition, emotion and pain. In the present study we investigated the effects of intra-agranular insular cortex microinjection of histamine and its H3 receptor agonist and antagonist on sensory and affective aspects of neuropathic pain. Spared nerve injury model of neuropathic pain was used. Two guide cannulas were surgically implanted in the right and left sides of agranular insular cortex. Sensory component (mechanical hyperalgesia) was recorded by application of von Frey filaments onto the plantar surface of the hind paw. Area under curve of mechanical hyperalgesia was calculated. Affective aspect (place escape avoidance paradigm) was recorded using an inverse white/black chamber. Histamine (0.5, 1 and 2 μg/site) and thioperamide (a histamine H3 receptor antagonist, 4 μg/site) decreased, whereas immepip (a histamine H3 receptor agonist, 2 μg/site) increased the percentages of paw withdrawal frequency and time spent in white side of white/black box. Prior administration of thioperamide (4 μg/site) increased the suppressive effects induced by histamine and inhibited immepip (2 μg/site)-induced hyperalgesia and aversion. Based on the present results, it is concluded that histamine and its H3 receptor at the agranular insular cortex level may involve in modulation of sensory and affective components of neuropathic pain.

Involvement of miR-34a and p53 protein of cerebral cortex in electroacpuncture analgesia in mice with neuropathic pain

To explore the involvement of miR-34a in cerebral cortex mediated anti-hyperalgesic effect of electroacupuncture (EA) in mice with neuropathic pain induced by chronic constriction injury (CCI) of sciatic nerve, so as to reveal its mechanisms underlying improvement of neuropathic pain. A total of 75 male C57BL/6 mice were equally randomized into 3 groups: sham, CCI model and CCI+EA (n=25 in each group). Mice of the sham group received simple separation of the right sciatic nerve without ligation. The CCI model was established by liagation of the right sciatic nerve. EA (2 Hz /15 Hz, 1 mA) was applied to bilateral "Zusanli" (ST36) and "Sanyinjiao" (SP9) for 30 min, once every other day. The mechanical and thermal pain threshold of the bilateral hind-paws was detected at the 3rd, 5th and 7th day after modeling, and the expression of miR-34a of bilateral cerebral cortex tissues and that of p53 protein of the left cerebral cortex were determined by using quantitive real time PCR and Western blot, respectively. The mechnical paw withdrawal frequency were significantly higher and the thermal paw withdrawal latencies (PWLs) were significantly shorter at the affected hind-limb (rather than at the healthy hind limb) on day 3, 5 and 7 in the CCI model group than those in the sham group (P<0.05), and considerably reversed at the affected hind-limb (rather than at the healthy hind limb) in the EA group than in the CCI model group (P<0.05), suggesting an analgesic effect of EA intervention. After modeling, the expression levels of miR-34a and p53 on day 3, 5 and 7 were significantly up-regulated in the left cerebral cortex tissue (rather than in the right cerebral cortex) of the CCI model group in comparison with the sham group (P<0.05). After EA intervention, the up-regulated expression levels of miR-34a and p53 in the left cerebral cortex tissue (rather than in the right cerebral cortex) were obviously suppressed in the EA group relevant to the CCI model group (P<0.05). EA stimulation of ST36 and SP9 can down-regulate the expression of miR-34a and p53 in the contra-lateral cerebral cortex tissue of the CCI mice, which may contribute to its anti-hyperalgesic effect.

Systemic Rapamycin Attenuates Morphine-Induced Analgesic Tolerance and Hyperalgesia in Mice.

Previous studies showed that repeated intrathecal morphine injection activated the mammalian target of rapamycin complex 1 (mTORC1) in spinal dorsal horn neurons and that blocking this activation by intrathecal infusion of rapamycin, a specific mTORC1 inhibitor, prevented the initiation of morphine-induced tolerance and hyperalgesia. However, in clinic, rapamycin is usually administrated orally. In this study, we examined whether systemic administration of rapamycin had the effect on morphine-induced tolerance and hyperalgesia in mice. Repeatedly intraperitoneal injection of morphine led to morphine analgesic tolerance on day 5 post-injection evidenced by a marked decrease in morphine's maximal possible analgesic effect and hyperalgesia on day 6 post-injection demonstrated by significant increases in paw withdrawal frequency in response to mechanical stimulation and decreases in paw withdrawal latency in response to cold stimulation on bilateral sides. Co-intraperitoneal injection with rapamycin prevented the development of morphine analgesic tolerance and hyperalgesia. Moreover, on day 6 after morphine injection, co-intraperitoneal injection with rapamycin reduced the established morphine tolerance and hyperalgesia. Co-intraperitoneal injection of rapamycin also attenuated the morphine-induced increases in the levels of phosphorylated mTOR and its downstream target phosphorylated 4E-BP1 in the spinal cord dorsal horn. Our findings indicate that, like intrathecal injection, systemic administration of rapamycin has significant effects on both induction and maintenance of morphine tolerance and hyperalgesia. Systemic mTOR inhibitors could serve as promising medications for use as adjuvants with opioids in clinical chronic pain management.

Islet-cell autoantigen 69 mediates the antihyperalgesic effects of electroacupuncture on inflammatory pain by regulating spinal glutamate receptor subunit 2 phosphorylation through protein interacting with C-kinase 1 in mice.

Electroacupuncture (EA) is widely used in clinical settings to reduce inflammatory pain. Islet-cell autoantigen 69 (ICA69) has been reported to regulate long-lasting hyperalgesia in mice. ICA69 knockout led to reduced protein interacting with C-kinase 1 (PICK1) expression and increased glutamate receptor subunit 2 (GluR2) phosphorylation at Ser880 in spinal dorsal horn. In this study, we evaluated the role of ICA69 in the antihyperalgesic effects of EA and the underlying mechanism through regulation of GluR2 and PICK1 in spinal dorsal horn. Hyperalgesia was induced in mice with subcutaneous plantar injection of complete Freund adjuvant (CFA) to cause inflammatory pain. Electroacupuncture was then applied for 30 minutes every other day after CFA injection. When compared with CFA group, paw withdrawal frequency of CFA+EA group was significantly decreased. Remarkable increases in Ica1 mRNA expression and ICA69 protein levels on the ipsilateral side were detected in the CFA+EA group. ICA69 expression reached the peak value around day 3. More importantly, ICA69 deletion impaired the antihyperalgesic effects of EA on GluR2-p, but PICK1 deletion could not. Injecting ICA69 peptide into the intrathecal space of ICA69-knockout mice mimicked the effects of EA analgesic and inhibited GluR2-p. Electroacupuncture had no effects on the total protein of PICK1 and GluR2. And, EA could increase the formation of ICA69-PICK1 complexes and decrease the amount of PICK1-GluR2 complexes. Our findings indicate that ICA69 mediates the antihyperalgesic effects of EA on CFA-induced inflammatory pain by regulating spinal GluR2 through PICK1 in mice.