Complete Freund's Adjuvant Model Research Articles

Shared nociceptive dorsal root ganglion neurons participating in acupoint sensitization.

When the body is under pathological stress (injury or disease), the status of associated acupoints changes, including decreased pain threshold. Such changes in acupoint from a “silent” to an “active” state are considered “acupoint sensitization,” which has become an important indicator of acupoint selection. However, the mechanism of acupoint sensitization remains unclear. In this study, by retrograde tracing, morphological, chemogenetic, and behavioral methods, we found there are some dorsal root ganglion (DRG) neurons innervating the ST36 acupoint and ipsilateral hind paw (IHP) plantar simultaneously. Inhibition of these shared neurons induced analgesia in the complete Freund’s adjuvant (CFA) pain model and obstruction of nociceptive sensation in normal mice, and elevated the mechanical pain threshold (MPT) of ST36 acupoint in the CFA model. Excitation of shared neurons induced pain and declined the MPT of ST36 acupoint. Furthermore, most of the shared DRG neurons express TRPV1, a marker of nociceptive neurons. These results indicate that the shared nociceptive DRG neurons participate in ST36 acupoint sensitization in CFA-induced chronic pain. This raised a neural mechanism of acupoint sensitization at the level of primary sensory transmission.

Cryoneurolysis with Injectable Ice Slurry Modulates Mechanical Skin Pain

Cutaneous pain is a common symptom of skin disease, and available therapies are inadequate. We developed a neural selective and injectable method of cryoneurolysis with ice slurry, which leads to a long-lasting decrease in mechanical pain. The aim of this study is to determine whether slurry injection reduces cutaneous pain without inducing the side effects associated with conventional cryoneurolysis. Using the rat sciatic nerve, we examined the effects of slurry on nerve structure and function in comparison with the effects of a Food and Drug Administration‒approved cryoneurolysis device (Iovera). Coherent anti-Stokes Raman scattering microscopy and immunofluorescence staining were used to investigate histological effects on the sciatic nerve and on downstream cutaneous nerve fibers. Complete Freund's Adjuvant model of cutaneous pain was used to study the effect of the slurry on reducing pain. Structural changes in myelin induced by slurry were comparable with those induced by Iovera, which uses much colder temperatures. Compared with that of Iovera, the decrease in mechanical pain due to slurry was less profound but lasted longer without signs of dysesthesia. Slurry did not cause a reduction of epidermal nerve fibers or a change in thermal pain sensitivity. Slurry-treated rats showed reduced cutaneous mechanical pain in response to Complete Freund's Adjuvant. Slurry injection can be used to successfully reduce cutaneous pain without causing dysesthesia.

Chymotrypsin attenuates adjuvant-induced arthritis by downregulating TLR4, NF-κB, MMP-1, TNF-α, IL-1β, and IL-6 expression in Sprague–Dawley rats

Objective Rheumatoid arthritis (RA) is mainly characterized by synovial hyperplasia, angiogenesis, inflammatory cells infiltration. Chymotrypsin is a proteolytic enzyme with anti-inflammatory effects. The current project was intended to test the efficacy and mechanism of chymotrypsin in adjuvant-induced arthritis (AIA) rats to provide an experimental basis for the clinical application of chymotrypsin. Methods Sprague–Dawley rats were injected with complete Freund’s adjuvant (CFA) in the hind left paw pad to establish an AIA model. Forty rats were randomly divided into five groups (n = 8): blank; CFA model (model); low-dose chymotrypsin (CLD), 0.53 mg/kg; high-dose chymotrypsin (CHD), 1.06 mg/kg; piroxicam, 10 mg/kg. The treatments were performed in the subplantar region of the left hind paw from Day 8 (D8) to Day 28 after adjuvant injection. The body weight, paw diameter, swelling degree of paw, and arthritic score were measured on D0, D7, D14, D21, and D28. All animals were sacrificed on D29. Subsequently, the synovial tissue of the ankle joint of the rats was stained with HE to generate pathological sections for observation of the pathological changes of synovial tissue from the ankle joint. The protein levels of MMP-1, TNF-α, IL-1β, and IL-6 in the rats’ serum were determined by ELISA. Western blotting was used to detect the protein expression of TLR4 and NF-κB in the rat ankle tissue. The mRNA expression of TLR4, NF-κB, IL-1β, IL-6, and TNF-α in synovial tissue of the ankle joint was detected by RT-qPCR. Results The body weight of the rats in each group showed an increasing trend, and there was no significant difference in weight between the groups. CHD and piroxicam suppressed paw swelling and arthritic scores and decreased synovial hyperplasia, inflammatory cell infiltration, pannus formation, and bone destruction. Furthermore, the overproduction of MMP-1, TNF-α, IL-1β, and IL-6 in serum was remarkably attenuated in the chymotrypsin- and piroxicam-treated rats. The protein levels of TLR4 and NF-κB in the synovial tissue of the chymotrypsin group and the piroxicam group were significantly lower than those in the model group. Likewise, the rats treated with chymotrypsin and piroxicam had a substantial decline in the mRNA expression of TLR4, NF-κB, TNF-α, IL-1β, and IL-6 in synovial tissue. Conclusions Chymotrypsin alleviates the joint damage of AIA rats, probably by reducing the expression of MMP-1, TNF-α, IL-1β, and IL-6 through TLR4/NF-κB signaling pathway.

Anterior Insular-nucleus Accumbens Pathway Controls Refeeding-induced Analgesia under Chronic Inflammatory Pain Condition

Feeding behaviors are closely associated with chronic pain in adult rodents. Our recent study revealed that 2 h refeeding after 24 h fasting (i.e., refeeding) attenuates pain behavior under chronic inflammatory pain conditions. However, while brain circuits mediating fasting-induced analgesia have been identified, the underlying mechanism of refeeding-induced analgesia is still elusive. Herein, we demonstrate that the neural activities in the nucleus accumbens shell (NAcS) and anterior insular cortex (aIC) were increased in a modified Complete Freund’s Adjuvant (CFA)-induced chronic inflammatory pain condition, which was reversed by refeeding. We also found that refeeding reduced the enhanced excitability of aICCaMKII–NAcSD2R projecting neurons in this CFA model. Besides, chemogenetic inhibition of aICCaMKII–NAcSD2R neural circuit suppressed chronic pain behavior while activation of this circuit reversed refeeding-induced analgesia. Thus, the present study suggests that aICCaMKII–NAcSD2R neural circuit mediates refeeding-induced analgesia, thereby serving as a potential therapeutic target to manage chronic pain.

Objective Quantification of Limb Position in Mouse Spared Nerve Injury Reveals New Motor Phenotypes

Difficulties translating basic animal research into novel pharmacotherapies for neuropathic pain can, in part, be attributed to a lack of predictive efficacy in preclinical pain models. Most animal models of neuropathic pain rely heavily on targeted nerve injuries quantified using reflexive measures in response to an applied noxious stimulus. However, data from these approaches fail to capture spontaneous and ongoing pain – a critical component of neuropathic pain in human patients. Furthermore, the common spared nerve injury (SNI) model damages the tibial and common peroneal nerves that should result in motor phenotypes not typically captured in preclinical literature. To overcome these limitations, we employed deep learning-based markerless pose estimation software (DeepLabCut) to quantify spontaneous limb position in C57BL/6J mice during a one-minute tail suspension following either SNI or sham surgery. Using this granular detail, we were able to characterize previously unidentified injury-induced motor behaviors that persist up to six weeks following SNI. In particular, we found a decrease in the median distance between body midline and injured limb position in the SNI group compared to sham controls. We also found some evidence for a decrease in toe movement relative to the heel movement. We repeated these analyses in subsequent cohorts of SNI mice to determine whether these outputs could be reversed by multiple analgesics with diverse mechanistic actions and thus far do not see evidence that these behaviors are reversed by analgesics. Furthermore, we do not see these phenotypes in the Complete Freund's Adjuvant model of inflammatory pain. Together these results suggest SNI causes previously unobserved motor phenotypes unrelated to altered sensation that are likely underappreciated while interpreting preclinical pain research. Grant support from R01 NS117899-01 F31 NS124301-01 Rita Allen Foundation.

Chronic facial inflammatory pain-induced anxiety is associated with bilateral deactivation of the rostral anterior cingulate cortex

Patients with chronic pain, especially orofacial pain, often suffer from affective disorders, including anxiety. Previous studies largely focused on the role of the caudal anterior cingulate cortex (cACC) in affective responses to pain, long-term potentiation (LTP) in cACC being thought to mediate the interaction between anxiety and chronic pain. But recent evidence indicates that the rostral ACC (rACC), too, is implicated in processing affective pain. However, whether such processing is associated with neuronal and/or synaptic plasticity is still unknown. We addressed this issue in a chronic facial inflammatory pain model (complete Freund's adjuvant model) in rats, by combining behavior, Fos protein immunochemistry and ex vivo intracellular recordings in rACC slices prepared from these animals. Facial mechanical allodynia occurs immediately after CFA injection, peaks at post-injection day 3 and progressively recovers until post-injection days 10–11, whereas anxiety is delayed, being present at post-injection day 10, when sensory hypersensitivity is relieved, but, notably, not at post-injection day 3. Fos expression reveals that neuronal activity follows a bi-phasic time course in bilateral rACC: first enhanced at post-injection day 3, it gets strongly depressed at post-injection day 10. Ex vivo recordings from lamina V pyramidal neurons, the rACC projecting neurons, show that both their intrinsic excitability and excitatory synaptic inputs have undergone long-term depression (LTD) at post-injection day 10. Thus chronic pain processing is associated with dynamic changes in rACC activity: first enhanced and subsequently decreased, at the time of anxiety-like behavior. Chronic pain-induced anxiety might thus result from a rACC deactivation-cACC hyperactivation interplay.

Behavioral Survey of Effects of Pulsed Radiofrequency on Neuropathic and Nociceptive Pain in Rats: Treatment Profile and Device Implantation

Pulsed radiofrequency (PRF) stimulation is widely used for intractable pain; however, there is no consensus on treatment protocols and appropriate types of pain. We compared effectiveness of bipolar and unipolar PRF on neuropathic or inflammatory pains, and of targets at the dorsal root ganglion (DRG) and sciatic nerve (SN). We also examined efficacy of repetitive PRF stimulations. This preclinical study could serve as an extensive survey before human trials. Spare nerve injury (SNI)-induced neuropathic pain and complete Freund's adjuvant (CFA) injection-induced inflammatory pain were used. Behavioral responses were measured using von Frey test, acetone test, and Hargreave's test at preinjury and postinjury time points. In both models, we evaluated results of DRG stimulation with unipolar PRF (45 V) versus bipolar PRF (5V), stimulation at DRG vs. SN, and repetitive stimulations. Both unipolar and bipolar PRFs reduced SNI- or CFA-induced pain for a similar duration. In the SNI model, PRF-DRG had a stronger effect on tactile pain than PRF-SN but lower effect on cold allodynia, whereas in the CFA model PRF-DRG and PRF-SN showed similar effects. Repetitive PRF stimulation, by open technique or implantation method, produced analogous effect by each stimulus, and no evident analgesic tolerance or neurological deficit was shown. PRF temporarily attenuates neuropathic and inflammatory pain. Bipolar PRF generates significant analgesia with a much lower electrical power than unipolar PRF. Meanwhile, the minor variant effects between PRF-DRG and PRF-SN may indicate distinct mechanisms. The sustained-analgesia by repetitive treatments suggests implantation technique could be a promising choice.

Tyrosine kinase type A-specific signalling pathways are critical for mechanical allodynia development and bone alterations in a mouse model of rheumatoid arthritis.

Rheumatoid arthritis is frequently associated with chronic pain that still remains difficult to treat. Targeting nerve growth factor (NGF) seems very effective to reduce pain in at least osteoarthritis and chronic low back pain but leads to some potential adverse events. Our aim was to better understand the involvement of the intracellular signalling pathways activated by NGF through its specific tyrosine kinase type A (TrkA) receptor in the pathophysiology of rheumatoid arthritis using the complete Freund adjuvant model in our knock-in TrkA/C mice. Our multimodal study demonstrated that knock-in TrkA/C mice exhibited a specific decrease of mechanical allodynia, weight-bearing deficit, peptidergic (CGRP+) and sympathetic (TH+) peripheral nerve sprouting in the joints, a reduction in osteoclast activity and bone resorption markers, and a decrease of CD68-positive cells in the joint with no apparent changes in joint inflammation compared with wild-type mice after arthritis. Finally, transcriptomic analysis shows several differences in dorsal root ganglion mRNA expression of putative mechanotransducers, such as acid-sensing ionic channel 3 and TWIK-related arachidonic acid activated K+ channel, as well as intracellular pathways, such as c-Jun, in the joint or dorsal root ganglia. These results suggest that TrkA-specific intracellular signalling pathways are specifically involved in mechanical hypersensitivity and bone alterations after arthritis using TrkA/C mice.

Inconsistent Time-Dependent Effects of Tetramethylpyrazine on Primary Neurological Disorders and Psychiatric Comorbidities.

The aim of this study was to investigate the time dependent effects of tetramethylpyrazine (TMP, main activity compound of Ligusticum chuanxiong Hort) on two neurological disorders and their neuropsychiatric comorbidities. 6 Hz corneal rapid kindling was used to induce epileptogenesis and the inflammatory pain was induced by intra-articular Complete Freund’s adjuvant (CFA) injection. The mechanical pain thresholds were measured using von Frey hair (D4, D11, D18, D25 after CFA first injection), and the vertical rearings of the mice was observed. To test the neuropsychiatric comorbidities, anxiety-like behaviors of mice were examined by open field and elevated plus maze tests. Two behavioral despair models, tail suspension test and forced swimming test were also used to evaluate the depressive like behaviors. The results showed that TMP administered from the initial day (D1-D35 in kindling model, D0-D14 and D0-D28 in CFA model) of modeling retarded both the developments of 6 Hz corneal rapid kindling epileptogenesis and the CFA induced inflammatory pain. In comparison, late periods administration of TMP (D21-D35 in kindling and D14-D28 in CFA model) showed no effect on the epileptogenesis and the generalized seizures (GS) of kindling, but alleviated maintenance of CFA induced inflammatory pain. Furthermore, we also found all TMP treatments from the initial day of modeling alleviated the co-morbid depressive and anxiety-like behaviors in both models; however, late periods treatments did not, either in kindling or the CFA induced inflammatory pain. BDNF/ERK signaling impairment was also tested by western blot, and the results showed that TMP administered from the initial day of modeling increased the hippocampal BDNF/ERK expression, whereas late period administration showed no effects. Overall, our findings reveal the inconsistent time dependent effects of Tetramethylpyrazine on neurological disorders and their relative neuropsychiatric comorbidities, and provide novel insight into the early application of TMP that might enhance hippocampal BDNF/ERK signaling to alleviate neuropsychiatric comorbidities in neurological diseases.

Anti-inflammatory properties of ethanolic extract from Vatairea macrocarpa leaves

Ethnopharmacological relevanceIn Brazilian traditional folk medicine, the leaves and heartwood from Vatairea macrocarpa (Benth) Ducke (Angelim-of-Cerrado) (Fabaceae family) are used as remedy after cold maceration for the treatment of the condition popularly known as rheumatism, as well as for the general inflammatory aspects such as pains, injury and swelling. The rheumatological and rheumatic diseases affect 0.3–1.0% of the world population and all long-term treatment with conventional medications lead to adverse effects. Aim of the studyTo investigate the chemical composition and the anti-inflammatory properties and of the ethanolic extract from V. macrocarpa leaves (EEVM) in experimental models. Material and methodsEEVM was chemically analyzed by spectrophotometry and the compounds characterization was performed by nuclear magnetic resonance and mass spectrometry. EEVM was evaluated in methylthiazolyldiphenyl-tetrazolium bromide (MTT) (3, 10, 30, and 90 μg/ml) and neutrophils phagocytic activity (1, 3, and 10 μg/ml) tests. For in vivo models, the EEVM (10, 30, 100, and 300 mg/kg) was orally administered (p.o.) for inflammatory evaluation in carrageenan-induced pleurisy in Swiss mice. The EEVM (30 and 100 mg/kg, p.o.) was tested against the Complete Freund Adjuvant (CFA)-induced paw persistent inflammation and Mycobacterium bovis (bacillus Calmette-Guerin - BCG)-induced pleurisy in C57bL6 mice. ResultsThe chemical composition of EEVM showed 157.06 mg (GAE)/g in relation to phenolic compounds, 82.13 mg (RUE)/g in relation to flavonoids and 48.99 mg (TAE)/g in relation to tannins. The flavonoid compounds identified were catechin, epicatechin and kaempferol-3-O-a-l-rhamnopyranoside. EEVM did not present cytotoxicity in MTT assay, however EEVM reduced phagocytic neutrophils activity at all tested concentration. EEVM significantly inhibited leukocytes migration/proteins exudation in carrageenan-induced pleurisy model. The daily administration of EEVM inhibited the inflammatory parameters in BCG and CFA models. ConclusionsThe present study showed anti-inflammatory features of EEVM (V. macrocarpa) as a natural agent against inflammatory diseases.

The T-type calcium channel antagonist, Z944, reduces spinal excitability and pain hypersensitivity.

Background and PurposeT‐type voltage‐gated calcium channels are an emerging therapeutic target for neurological disorders including epilepsy and pain. Inhibition of T‐type channels reduces the excitability of peripheral nociceptive sensory neurons and reverses pain hypersensitivity in male rodent pain models. However, administration of peripherally restricted T‐type antagonists failed to show efficacy in multiple clinical and preclinical pain trials, suggesting that inhibition of peripheral T‐type channels alone may be insufficient for pain relief.Experimental ApproachWe utilized the selective and CNS‐penetrant T‐type channel antagonist, Z944, in electrophysiological, calcium imaging and behavioural paradigms to determine its effect on lamina I neuron excitability and inflammatory pain behaviours.Key ResultsVoltage‐clamp recordings from lamina I spinal neurons of adult rats revealed that approximately 80% of neurons possess a low threshold T‐type current, which was blocked by Z944. Due to this highly prevalent T‐type current, Z944 potently blocked action‐potential evoked somatic and dendritic calcium transients in lamina I neurons. Moreover, application of Z944 to spinal cord slices attenuated action potential firing rates in over half of laminae I/II neurons. Finally, we found that intraperitoneal injection of Z944 (1–10 mg·kg−1) dose‐dependently reversed mechanical allodynia in the complete Freund's adjuvant model of persistent inflammatory pain, with a similar magnitude and time course of analgesic effects between male and female rats.Conclusion and ImplicationsT‐type calcium channels critically shape the excitability of lamina I pain processing neurons and inhibition of these channels by the clinical stage antagonist Z944 potently reverses pain hypersensitivity across sexes.

Peripheral signaling mechanisms underlying mu‐delta opioid synergy

In response to the ongoing opioid epidemic, and in an effort to develop new analgesics with improved safety profiles, researchers have invested significant energy into further understanding the various downstream signaling cascades initiated by opioid receptor activation. Recent studies have demonstrated that G protein-biased ligands demonstrate reduced side effects such as respiratory depression and tolerance. Further research has shown that bivalent or bifunctional opioid compounds can also result in differential signal transduction. We hypothesized that a combination of mu and delta opioid agonists would engage different signaling pathways than drugs that target only one opioid receptor. Therefore, we probed the signaling mechanisms underlying the behavioral analgesic synergy previously demonstrated by a 1:1 combination of loperamide, a peripherally-restricted mu-opioid agonist, and oxymorphindole, a delta-opioid agonist. Using the complete Freund's adjuvant model of inflammatory pain and the Hargreaves assay for thermal nociception, the anti-hyperalgesic effect of loperamide-oxymorphindole in male and female mice was challenged by co-administration of a recently developed antagonist against MOR-DOR heteromers, D24M. Then, mice were given an injection of loperamide, oxymorphindole, or their combination with and without pertussis toxin in order to assess the involvement of Gai/o signaling on the behavioral anti-hyperalgesia. Finally, in naïve mice, the role of G protein-coupled inwardly-rectifying potassium (GIRK) channels, a downstream target of G proteins, was investigated using the peptide inhibitor tertiapin-Q, as well as GIRK KO mice. The data demonstrate that D24M was significantly more potent in antagonizing the loperamide-oxymorphindole combination (p<0.01) than either drug alone, and that pertussis toxin significantly reduces the anti-hyperalgesic effect of the combination (p<0.05). Finally, both pharmacologic and genomic inhibition of GIRK2 function results in a significant reduction of the combination's anti-nociceptive effect (p<0.0001). From these data we conclude that the loperamide-oxymorphindole combination binds to heterodimeric opioid receptors, activates a G protein signaling pathway, and opens GIRK channels.

Naloxone-induced analgesia mediated by central kappa opioid system in chronic inflammatory pain

Opioids, which are widely used for the treatment of chronic pain, have an analgesic effect by mainly activating mu-opioid receptor (MOR). Paradoxically, a high dose of naloxone, non-selective opioid receptor antagonist, is also known to induce analgesia, but the underlying mechanism remains unclear. Since kappa-opioid receptor (KOR) and dynorphin (KOR ligand) have been implicated in the naloxone-induced analgesia, we aimed to elucidate its mechanism by focusing on the kappa-opioid system in the brain under inflammatory pain condition. Systemic administration of naloxone (10 mg/kg, i.p.) decreased spontaneous pain behaviors only in complete Freund's adjuvant (CFA)-induced chronic inflammatory pain model but not in the formalin-induced acute pain model. Immunohistochemistry analysis in the CFA model revealed both a significant decrease in MOR expression and an increase in prodynorphin density in the central nucleus of theamygdala (CeA) and nucleus accumbens (NAc) but not in other brain areas. Systemic administration of KOR antagonist (norbinaltorphimine, nor-BNI 10 mg/kg) also decreased spontaneous pain behaviors in the CFA model. Furthermore, microinjection of both naloxone and nor-BNI into NAc and CeA significantly reduced spontaneous chronic pain behavior. Taken together, our results suggest that naloxone-induced analgesia may be mediated by blocking facilitated kappa-opioid systems in the NAc and CeA.

Anti-inflammatory Properties of Ethanolic Extract and 2″-O-β-d-Glucopyranosyl-vitexin Obtained from Alternanthera tenella Colla Whole Plant

Six flavonoids were identified and isolated from the ethanolic extract of Alternanthera tenella Colla (Amaranthaceae) whole plant (EEAT) including 2″-O-β-D-glucopyranosyl-vitexin (A19). Flavonoids have anti-inflammatory activity; however, the 2″-O-β-D-glucopyranosyl-vitexin anti-inflammatory property was not totally explored. The aim of the present study was to investigate the anti-inflammatory effects of ethanolic extract from A. tenella whole plant and isolated flavone C-glycoside A19 in models of inflammation. The oral administration (p.o.) of EEAT (30, 100, and 300 mg/kg), A19 (0.1, 1, and 10 mg/kg), and prednisolone (3 mg/kg) were tested against the carrageenan-induced paw edema in Swiss mice. The EEAT (100 mg/kg, p.o.), A19 (1 mg/kg, p.o.), and prednisolone (3 mg/kg, p.o.) were tested in the zymosan-articular inflammation, carrageenan-pleurisy, and complete Freund's adjuvant (CFA) models in Swiss mice. In silico analysis and search for structural relationships between vitexin derivatives flavones present in the EEAT and TNF-α inhibitors were performed. EEAT, A19, and prednisolone significantly inhibited (i) edema, mechanical hyperalgesia in carrageenan-induced paw inflammation; (ii) leukocyte migration and protein extravasation in carrageenan-induced pleurisy; (iii) knee edema, mechanical hyperalgesia, and leukocyte migration in articular inflammation induced by zymosan. Still the CFA induced the increase in myeloperoxidase and N-acetylglucosaminidase activities, EEAT, A19, and prednisolone significantly inhibited these enzymes. The in silico analysis showed that molecules with similar structure to apigenin and vitexin have a potential inhibition on the TNF system. This study confirms the anti-inflammatory properties of EEAT and A19. The C-glycosylated flavone A19 could be responsible for the EEAT anti-edematogenic and anti-hyperalgesic effects and a potential TNF-α inhibitor.

Therapeutic potential of russelioside B as anti-arthritic agent in Freund's adjuvant-induced arthritis in rats

Ethnopharmacological relevanceCaralluma species are traditional edible herbs used in folkloric medicine as antidiabetic, antioxidant, antipyretic, antirheumatic, anti-inflammatory and anthelmintic agents. C. quadrangula was selected in this study to document the traditional use of the genus as anti-rheumatic treatment and the possible mechanisms of action. Aim of the studyThe higher mortality rates and shorter survival among the patients suffering from rheumatoid arthritis (RA) led to the increased interest on searching for new treatments for RA. Russelioside B (RB), a major pregnane glycoside found in C. quadrangula, was evaluated as a new anti-rheumatic agent. Materials and methodsThe n-butanol fraction of C. quadrangula was chromatographed on a silica gel column to isolate RB. The adjuvant-induced arthritis (AIA) model was established in rats by intradermal injection of complete Freund's adjuvant (CFA) to evaluate its anti-arthritic effect. Ibuprofen was used as a reference drug. Forty rats were randomly divided into 5 groups (n = 8): normal (NOR); CFA model (CFA); ibuprofen, 5 mg/kg; RB, 25 mg/kg and RB, 50 mg/kg. The treatments were initiated from day 16 when AIA model was established and continued up to day 40. Serum diagnostic rheumatoid markers, inflammatory cytokines, oxidative stress biomarkers, cartilage and bone degeneration enzymes were assessed. ResultsRB at 50 mg/kg b. wt., showed significant decreases in the activities of hyaluronidase and β-glucouronidase enzymes as well significant decreases in the levels of proinflammatory cytokines as nuclear factor-kappa-B (NF-κB), tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) compared to the CFA group; 11.04 ± 0.61 pg/mg protein, 4.35 ± 0.25 pg/mg protein, 3.32 ± 0.13 pg/mg protein & 2.75 ± 0.14 pg/mg protein for RB, 50 mg/kg b. wt. group vs. 25.33 ± 2.13 pg/mg protein, 25.65 ± 2.1 pg/mg protein, 22.20 ± 1.34 pg/mg protein & 13.27 ± 1.40 pg/mg protein for the arthritic group, respectively. The total antioxidant capacity (TAC) was significantly restored to normal values in RB, 50 mg/kg treated rats (4.01 ± 0.09 nmol/mL vs. 3.71 ± 0.27 nmol/mL) and the levels of myeloperoxidase (MPO) reduced by 10-folds of the CFA arthritic group. Bone histomorphometry revealed that RB treatment significantly attenuated the CFA-induced bone loss in a dose-dependent manner. ConclusionsThese findings suggested that the anti-arthritic effect of RB was mediated through the reduction of the rheumatoid markers, anti-inflammatory and antioxidant action, inhibition of cartilage and bone degenerative enzymes as well as attenuation of bone loss and osteoclastogenesis.

The interaction between P2X3 and TRPV1 in the dorsal root ganglia of adult rats with different pathological pains

Peripheral inflammatory and neuropathic pain are closely related to the activation of purinergic receptor P2X ligand-gated ion channel 3 (P2X3) and transient receptor potential vanilloid 1 (TRPV1), but the interaction between P2X3 and TRPV1 in different types of pathological pain has rarely been reported. In this study, complete Freund’s adjuvant (CFA)-induced inflammatory pain and spared nerve injury (SNI)-induced neuropathic pain models were established in adult rats. The interactions between P2X3 and TRPV1 in the dorsal root ganglion were observed by pharmacological, co-immunoprecipitation, immunofluorescence and whole-cell patch-clamp recording assays. TRPV1 was shown to promote the induction of spontaneous pain caused by P2X3 in the SNI model, but the induction of spontaneous pain behaviour by TRPV1 was not completely dependent on P2X3 in vivo. In both the CFA and SNI models, the activation of peripheral P2X3 enhanced the effect of TRPV1 on spontaneous pain, while the inhibition of peripheral TRPV1 reduced the induction of spontaneous pain by P2X3 in the CFA model. TRPV1 and P2X3 had inhibitory effects on each other in the inflammatory pain model. During neuropathic pain, P2X3 facilitated the function of TRPV1, while TRPV1 had an inhibitory effect on P2X3. These results suggest that the mutual effects of P2X3 and TRPV1 differ in cases of inflammatory and neuropathic pain in rats.

Morphine Antinociception Restored by Use of Methadone in the Morphine-Resistant Inflammatory Pain State.

The antinociceptive effect of methadone in the morphine-resistant inflammatory pain state was described in the paw-withdrawal test using the complete Freund’s adjuvant (CFA)-induced mouse inflammatory pain model. After intraplantar (i.pl.) injection of CFA, thermal hyperalgesia was observed in the ipsilateral paw. The antinociceptive effects of subcutaneous (s.c.) injection of morphine, fentanyl, and oxycodone against thermal hyperalgesia in the inflammatory pain state were reduced in the ipsilateral paw 7 days after CFA pretreatment. On the contrary, the antinociceptive effect of s.c. injection of methadone was maintained in the ipsilateral paw 7 days after CFA pretreatment. The suppressed morphine antinociception in the CFA model mice was bilaterally restored following s.c. treatment with methadone 20 min prior to or 3 days after CFA pretreatment. The suppressed morphine antinociception was also bilaterally restored by intraperitoneal treatment with MK-801 30 min prior to CFA pretreatment; however, the s.c. injection of morphine 30 min prior to CFA pretreatment failed to restore the suppressed morphine antinociception in the CFA model mice. The expression level of mRNA for µ-opioid receptors 7 days after i.pl. pretreatment was not significantly changed by i.pl. pretreatment with CFA or s.c. pretreatment with methadone. In conclusion, methadone is extremely effective against thermal hyperalgesia in the morphine-resistant inflammatory pain state, and restores suppressed morphine antinociception in the inflammatory pain state without altering the expression level of mRNA for µ-opioid receptors.

Activation of HDAC4 and GR signaling contributes to stress-induced hyperalgesia in the medial prefrontal cortex of rats

“Stress-induced hyperalgesia (SIH)” is a phenomenon that stress can lead to an increase in pain sensitivity. Epigenetic mechanisms have been known to play fundamental roles in stress and pain. Histone acetylation is an epigenetic feature that is changed in numerous stress-related disease situations. However, epigenetic mechanism for SIH is not well known. We investigated the effect of histone acetylation on pain hypersensitivity using SPS (single-prolonged stress) + CFA (complete Freund’s adjuvant) model. We showed that the glucocorticoid receptor (GR)-pERK-pCREB-Fos signaling pathway was upregulated on stress-induced hyperalgesia and the paw withdrawal threshold in the SPS + CFA group dropped significantly compared with the SPS or CFA group. Histone deacetylases 4 (HDAC4)-expressing neurons in the medial prefrontal cortex (mPFC) were increased in the SPS + CFA-exposed group compared with CFA-exposed or SPS-exposed group. And we showed that the effects of stress-induced hyperalgesia were critically regulated via reversible acetylation (HDAC4) of the GR. Inhibiting HDAC4 by microinjection of sodium butyrate into the mPFC could disrupt glucocorticoid receptor (GR) signaling pathway, which lowered SPS + CFA-caused mechanical allodynia and alleviated anxiety-like behavior. Together, our studies suggest that HDAC inhibitors might involve in the process of stress-induced hyperalgesia.

Alterations in SUMOylation of the hyperpolarization-activated cyclic nucleotide-gated ion channel 2 during persistent inflammation.

BackgroundUnilateral injection of Complete Freund's Adjuvant (CFA) into the intra‐plantar surface of the rodent hindpaw elicits chronic inflammation and hyperalgesia in the ipsilateral hindlimb. Mechanisms contributing to this hyperalgesia may act over multiple time courses and can include changes in ion channel expression and post‐translational SUMOylation. Hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels mediate the hyperpolarization‐activated current, Ih. An HCN2‐mediated increase in C‐nociceptor Ih contributes to mechanical hyperalgesia in the CFA model of inflammatory pain. Changes in HCN2 post‐translational SUMOylation and protein expression have not been systematically documented for a given dorsal root ganglia (DRG) throughout the time course of inflammation.MethodsThis study examined HCN2 protein expression and post‐translational SUMOylation in a rat model of CFA‐induced hindpaw inflammation. L5 DRG cryosections were used in immunohistochemistry experiments and proximity ligation assays to investigate HCN2 expression and SUMOylation, respectively, on days 1 and 3 post‐CFA.ResultsUnilateral CFA injection elicited a significant bilateral increase in HCN2 staining intensity in small diameter DRG neurons on day 1 post‐CFA, and a significant bilateral increase in the number of small neurons expressing HCN2 but not staining intensity on day 3 post‐CFA. HCN2 channels were hyper‐SUMOylated in small diameter neurons of ipsilateral relative to contralateral DRG on days 1 and 3 post‐CFA.ConclusionsUnilateral CFA injection elicits unilateral mechanical hyperalgesia, a bilateral increase in HCN2 expression and a unilateral increase in post‐translational SUMOylation. This suggests that enhanced HCN2 expression in L5 DRG is not sufficient for mechanical hyperalgesia in the early stages of inflammation and that hyper‐SUMOylation of HCN2 channels may also be necessary.SignificanceNociceptor HCN2 channels mediate an increase in Ih that is necessary for mechanical hyperalgesia in a CFA model of chronic pain, but the mechanisms producing the increase in nociceptor Ih have not been resolved. The data presented here suggest that the increase in Ih during the early stages of inflammation may be mediated by an increase in HCN2 protein expression and post‐translational SUMOylation.

Preparation and characterization of C-phycocyanin coated with STMP/STPP cross-linked starches from different botanical sources

This work aimed to use sodium trimetaphosphate/sodium tripolyphosphate cross-linked potato, banana, corn, cassava, and breadfruit starches as wall materials for C-phycocyanin encapsulation, characterize them and evaluate their in vivo pharmacological effects in an inflammation model. The cross-linked starches were successfully obtained, characterized, and submitted to C-phycocyanin encapsulation by freeze-drying. The characterization of cross-linked starches-C-phycocyanin composites by scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetry, and differential scanning calorimetry demonstrated that the C-phycocyanin was encapsulated between amorphous chains of cross-linked starches. Among the five preparations, the cross-linked potato starch presented the highest phosphorous content (0.084%), substitution degree (0.004), water uptake capacity (0.88 g g−1), and C-phycocyanin encapsulation efficiency (67.58%), thus was tested in vivo. The cross-linked potato starch-C-phycocyanin prolonged the antihyperalgesic effects attributed to C-phycocyanin, evaluated by complete Freund's adjuvant (CFA) model. Starch cross-linking promoted the formation of a hydrogel network in swollen state entrapping C-phycocyanin, thus, acting as a barrier to its release to the medium and promoting long-lasting in vivo effects. The combination of chemical modification of starches followed by physical treatment presented itself as a useful tool for the development of pharmaceutical formulations.