Mouse Model Of Inflammatory Pain Research Articles

Differential alternation of the antinociceptive effect of narcotic analgesics on the inflammatory pain state

Antinociceptive effect of narcotic analgesics, fentanyl, oxycodone and methadone in inflammatory pain state was described in the von Frey filament test using the complete Freund's adjuvant (CFA)-induced mouse inflammatory pain model. After the i.pl. injection of CFA, mechanical allodynia (decrease of mechanical threshold) was observed in ipsilateral paw. The antinociceptive effect of fentanyl and oxycodone injected s.c. against mechanical allodynia in inflammatory pain state was reduced bilaterally at 1 day after CFA pretreatment. However, the antinociceptive effect of methadone injected s.c. against mechanical allodynia in inflammatory pain state was reduced unilaterally at 1 day after CFA pretreatment. Moreover, the reduction of the antinociceptive effect of methadone in ipsilateral paw on inflammatory pain state was smaller than those of fentanyl or oxycodone. In conclusion, the alternation of the antinociceptive effect of narcotic analgesics in inflammatory pain state is variable among the narcotic analgesics used.

Secretion and N-Linked Glycosylation Are Required for Prostatic Acid Phosphatase Catalytic and Antinociceptive Activity

Secretory human prostatic acid phosphatase (hPAP) is glycosylated at three asparagine residues (N62, N188, N301) and has potent antinociceptive effects when administered to mice. Currently, it is unknown if these N-linked residues are required for hPAP protein stability and activity in vitro or in animal models of chronic pain. Here, we expressed wild-type hPAP and a series of Asn to Gln point mutations in the yeast Pichia pastoris X33 then analyzed protein levels and enzyme activity in cell lysates and in conditioned media. Pichia secreted wild-type recombinant (r)-hPAP into the media (6–7 mg protein/L). This protein was as active as native hPAP in biochemical assays and in mouse models of inflammatory pain and neuropathic pain. In contrast, the N62Q and N188Q single mutants and the N62Q, N188Q double mutant were expressed at lower levels and were less active than wild-type r-hPAP. The purified N62Q, N188Q double mutant protein was also 1.9 fold less active in vivo. The N301Q mutant was not expressed, suggesting a critical role for this residue in protein stability. To explicitly test the importance of secretion, a construct lacking the signal peptide of hPAP was expressed in Pichia and assayed. This “cellular” construct was not expressed at levels detectable by western blotting. Taken together, these data indicate that secretion and post-translational carbohydrate modifications are required for PAP protein stability and catalytic activity. Moreover, our findings indicate that recombinant hPAP can be produced in Pichia—a yeast strain that is used to generate biologics for therapeutic purposes.

Attenuation of TRPV1 and TRPV4 Expression and Function in Mouse Inflammatory Pain Models Using Electroacupuncture

Although pain is a major human affliction, our understanding of pain mechanisms is limited. TRPV1 (transient receptor potential vanilloid subtype 1) and TRPV4 are two crucial receptors involved in inflammatory pain, but their roles in EA- (electroacupuncture-) mediated analgesia are unknown. We injected mice with carrageenan (carra) or a complete Freund's adjuvant (CFA) to model inflammatory pain and investigated the analgesic effect of EA using animal behavior tests, immunostaining, Western blotting, and a whole-cell recording technique. The inflammatory pain model mice developed both mechanical and thermal hyperalgesia. Notably, EA at the ST36 acupoint reversed these phenomena, indicating its curative effect in inflammatory pain. The protein levels of TRPV1 and TRPV4 in DRG (dorsal root ganglion) neurons were both increased at day 4 after the initiation of inflammatory pain and were attenuated by EA, as demonstrated by immunostaining and Western blot analysis. We verified DRG electrophysiological properties to confirm that EA ameliorated peripheral nerve hyperexcitation. Our results indicated that the AP (action potential) threshold, rise time, and fall time, and the percentage and amplitude of TRPV1 and TRPV4 were altered by EA, indicating that EA has an antinociceptive role in inflammatory pain. Our results demonstrate a novel role for EA in regulating TRPV1 and TRPV4 protein expression and nerve excitation in mouse inflammatory pain models.

The identification, and optimisation of hERG selectivity, of a mixed NET/SERT re-uptake inhibitor for the treatment of pain

Hit compound 1, a selective noradrenaline re-uptake transporter (NET) inhibitor was optimised to build in potency at the serotonin re-uptake transporter (SERT) whilst maintaining selectivity against the dopamine re-uptake transporter (DAT). During the optimisation of 1 it became clear that selectivity against the Kv11.1 potassium ion channel (hERG) was also a parameter for optimisation within the series. Discrete structural changes to the molecule as well as a lowering of global cLogP successfully increased the hERG selectivity to afford compound 11m, which was efficacious in a mouse model of inflammatory pain, complete Freund’s adjuvant (CFA) induced thermal hyperalgesia and a rat model of neuropathic pain, spinal nerve ligation (SNL) induced mechanical allodynia.

Regulation of Inflammatory Pain by Inhibition of Fatty Acid Amide Hydrolase

Although cannabinoids are efficacious in laboratory animal models of inflammatory pain, their established cannabimimetic actions diminish enthusiasm for their therapeutic development. Conversely, fatty acid amide hydrolase (FAAH), the chief catabolic enzyme regulating the endogenous cannabinoid N-arachidonoylethanolamine (anandamide), has emerged as an attractive target for treating pain and other conditions. Here, we tested WIN 55212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de)-1,4-benzoxazin-6-yl]-1-napthalenylmethanone], a cannabinoid receptor agonist, and genetic deletion or pharmacological inhibition of FAAH in the lipopolysaccharide (LPS) mouse model of inflammatory pain. WIN 55212-2 significantly reduced edema and hot-plate hyperalgesia caused by LPS infusion into the hind paws, although the mice also displayed analgesia and other central nervous system effects. FAAH(-/-) mice exhibited reduced paw edema and hyperalgesia in this model without apparent cannabimimetic effects. Transgenic mice expressing FAAH exclusively on neurons continued to display the antiedematous, but not the antihyperalgesic, phenotype. The CB(2) cannabinoid receptor (CB(2)) antagonist SR144528 [N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide] blocked this non-neuronal, anti-inflammatory phenotype, and the CB(1) cannabinoid receptor (CB(1)) antagonist rimonabant [SR141716, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] blocked the antihyperalgesic phenotype. The FAAH inhibitor URB597 [cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester] attenuated the development of LPS-induced paw edema and reversed LPS-induced hyperalgesia through the respective CB(2) and CB(1) mechanisms of action. However, the transient receptor potential vanilloid type 1 antagonist capsazepine did not affect either the antihyperalgesic or antiedematous effects of URB597. Finally, URB597 attenuated levels of the proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha in LPS-treated paws. These findings demonstrate that simultaneous elevations in non-neuronal and neuronal endocannabinoid signaling are possible through inhibition of a single enzymatic target, thereby offering a potentially powerful strategy for treating chronic inflammatory pain syndromes that operate at multiple levels of anatomical integration.

Endogenous N-Acetylaspartylglutamate (NAAG) Inhibits Synaptic Plasticity/Transmission in the Amygdala in a Mouse Inflammatory Pain Model

BackgroundThe peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is widely expressed throughout the vertebrate nervous system, including the pain processing neuraxis. Inhibitors of NAAG peptidases are analgesic in animal models of pain. However, the brain regions involved in NAAG's analgesic action have not been rigorously defined. Group II metabotropic glutamate receptors (mGluR2/3) play a role in pain processing in the laterocapsular part of the central nucleus of the amygdala (CeLC). Given the high concentration of NAAG in the amygdala and its activation of group II mGluRs (mGluR3 > mGluR2), this study was undertaken using the mouse formalin model of inflammatory pain to test the hypothesis that NAAG influences pain processing in the amygdala. Evoked excitatory postsynaptic currents (eEPSCs) were studied in neurons in the CeLC of mouse brain slices following stimulation of the spinoparabrachial amygdaloid afferents.ResultsApplication of a NAAG peptidase inhibitor, ZJ43, dose dependently inhibited the amplitude of the eEPSCs by up to 50% in control CeLC demonstrating the role of NAAG in regulation of excitatory transmission at this synapse. A group II mGluR agonist (SLx-3095-1) similarly inhibited eEPSC amplitude by about 30%. Both effects were blocked by the group II mGluR antagonist LY341495. ZJ43 was much less effective than SLx in reducing eEPSCs 24 hours post inflammation suggesting an inflammation induced reduction in NAAG release or an increase in the ratio of mGluR2 to mGluR3 expression. Systemic injection of ZJ43 proximal to the time of inflammation blocked peripheral inflammation-induced increases in synaptic transmission of this pathway 24 hrs later and blocked the induction of mechanical allodynia that developed by this time point.ConclusionsThe main finding of this study is that NAAG and NAAG peptidase inhibition reduce excitatory neurotransmission and inflammation-induced plasticity at the spinoparabrachial synapse within the pain processing pathway of the central amygdaloid nucleus.

Reflecting on tragedy: A commentary on deaths of children in restraints

Paracetamol (acetaminophen) is the most commonly used analgesic in the world. Recently, a new view of its action has emerged: that paracetamol would be a pro-drug that should be metabolized by the FAAH enzyme into AM404, its active metabolite. However, this hypothesis has been demonstrated only in naive animals, a far cry from the clinical pathologic context of paracetamol use. Moreover, FAAH is a ubiquitous enzyme expressed both in the central nervous system and in the periphery. Thus, we explored: (i) the involvement of FAAH in the analgesic action of paracetamol in a mouse model of inflammatory pain; and (ii) the contributions of central versus peripheral FAAH in this action. The analgesic effect of paracetamol was evaluated in thermal hyperalgesia, mechanical allodynia and hyperalgesia induced by an intra-plantar injection of carrageenan (3%) in FAAH knock-out mice or their littermates. Moreover, the contribution of the central and peripheral enzymes was explored by comparing the effect of a global FAAH inhibitor (URB597) to that of a peripherally restricted FAAH inhibitor (URB937) on paracetamol action. Here, we show that in a model of inflammatory pain submitted to different stimuli, the analgesic action of paracetamol was abolished when FAAH was genetically or pharmacologically inhibited. Whereas a global FAAH inhibitor, URB597 (0.3 mg/kg), reduced the anti-hyperalgesic action of paracetamol, a brain-impermeant FAAH inhibitor, URB937 (0.3 mg/kg), had no influence. However, administered intracerebroventricularly, URB937 (5 μg/mouse) reduced the action of paracetamol. These results demonstrate that the supra-spinally-located FAAH enzyme is necessary for the analgesic action of paracetamol.

The Additive Antinociceptive Interaction Between WIN 55,212-2, a Cannabinoid Agonist, and Ketorolac

Combinations of nonsteroidal antiinflammatory drugs (NSAIDs) and opioids are widespread in the management of pain, allowing better analgesia with reduced side effects. Cannabinoids are promising analgesic drugs that have pharmacological properties similar to those of opioids. However, the beneficial effects of cannabinoids for pain treatment are counterbalanced by their psychotomimetic side effects. We designed the present study to evaluate the antinociceptive interaction between cannabinoids and NSAIDs in mice, using the acetic acid-induced writhing test and tail-flick test. Interactions were analyzed using isobolographic analysis. WIN 55,212-2, a cannabinoid agonist, and the NSAID ketorolac, either alone or in combination, produced dose-dependent antinociception in the writhing test. Isobolographic analysis showed additive interactions between WIN 55,212-2 and ketorolac when they were coadministered systemically. Ketorolac is inactive in the radiant heat tail-flick test in which WIN 55,212-2 was active. Ketorolac did not influence WIN 55,212-2-induced antinociception in the tail-flick test. This study demonstrated an additive antinociceptive interaction between WIN 55,212-2 and ketorolac in an inflammatory visceral pain model. The combination of cannabinoids and NSAIDs may have utility in the pharmacotherapy of pain.