Animal Studies

Morphine is generally used to treat chronic pain in clinic. But long-term use of morphine can inevitably induce analgesic tolerance and hyperalgesia. Caveolin-1 is reported to affect morphine-mediated signaling transduction. However, the action mechanism of morphine-induced analgesic tolerance is still unknown. In this study, morphine-induced analgesic tolerance model was established in Sprague-Dawley rats. The effects of Caveolin-1 blocking on neuroinflammation and ERK/c-JUN pathway were then explored. Morphine can remarkably elevate the expression level of Caveolin-1. Based on paw withdrawal latency behavior test, we found that Caveolin-1 blocking can effectively attenuate morphine-induced analgesic tolerance and neuroinflammation. Activation of ERK/c-JUN significantly reversed the above influences caused by Caveolin-1 blocking. Taken together, blocking of Caveolin-1 can attenuate morphine-induced inflammation and analgesic tolerance through inhibiting NLRP3 inflammasome and ERK/c-JUN pathway.

Accumulation of uric acid (UA) during muscular trauma is a factor involved in the development of muscle hyperalgesia. Neutrophil extracellular traps (NETs), DNA-based reticular structures to capture UA, play a central role in the pain onset of gout attacks; however, the involvement of NETs via the elevation of local UA level in muscle hyperalgesia due to injuries from muscle overuse remains unknown. The triceps surae muscles (TSMs) in the unilateral hindlimb of mice were electrically stimulated to induce excessive muscle contraction. Mechanical withdrawal thresholds, tissue UA levels, neutrophil recruitment, and protein amount of citrullinated histone 3 (citH3), a major marker of NETs, were investigated. Furthermore, whether neutrophil depletion, extracellular DNA cleavage, and administration of the urate-lowering agent febuxostat improved muscle hyperalgesia caused by NET formation was examined. CitH3 expression upon neutrophil recruitment was significantly increased in the stimulated TSMs with increased tissue UA levels, whereas febuxostat administration improved muscle hyperalgesia with decreased citH3 and tissue UA levels, as observed in neutrophil depletion and extracellular DNA digestion. The underlying mechanism of muscle hyperalgesia associated with locally recruited neutrophils forming NETs due to increased tissue UA levels potentially plays a significant role in creating a vicious circle of muscle pain.

Mas-related G protein-coupled receptor D (MrgprD) was first identified in small-diameter sensory neurons of mouse dorsal root ganglion (DRG). The role of MrgprD has been studied in somatosensation, especially in pain and itch response. We recently showed that MrgprD also participated in the modulation of murine intestinal motility. The treatment of MrgprD receptor agonist suppressed the spontaneous contractions in the isolated intestinal rings of mice, indicating the intrinsic expression of MrgprD in the murine gastrointestinal (GI) tract. Although the expression of Mrgprd in GI tract has been previously detected by the way of quantitative real-time PCR, the cell-type-specific expression of MrgprD in GI tract is no yet determined. Herein, we employed Mrgprd-tdTomato reporter mouse line and the whole-mount immunohistochemistry to observe the localization of MrgprD in the smooth muscle layers of ileum and colon. We show that tdTomato-positive cells colocalized with NeuN-immunostaining in the myenteric plexus in the whole-mount preparations of the ileum and the colon. Further immunohistochemistry using the commercially available MrgprD antibody revealed the expression of MrgprD in NeuN-labeled enteric neurons in the myenteric plexus. Our results demonstrate the expression of MrgprD in the enteric neurons in the murine GI tract, highlighting the implications of MrgprD in the physiology and pathophysiology of the GI tract.

Painful diabetic neuropathy is one of the most common complications of diabetes in humans. The current treatments are not completely effective, and the main mechanisms implicated in the development of diabetic neuropathy are not completely elucidated. Thus, in male db/db mice, a murine model of type 2 diabetes, we investigated the effects of treatment with a heme oxygenase 1 (HO-1) inducer, cobalt protoporphyrin IX (CoPP), on the 1) hyperglycemia and mechanical allodynia associated with type 2 diabetes and 2) molecular changes induced by diabetic neuropathy in the central nervous system (CNS). Thus, we evaluated the effects of CoPP on the protein levels of 4-HNE (oxidative stress), Nrf2, superoxide dismutase 1 (SOD1), NAD(P)H quinone oxidoreductase 1 (NQO1), HO-1, glutathione S-transferase Mu 1 (GSTM1) (antioxidant enzymes), phosphatidylinositol 3-kinase/protein kinase B (nociceptive pathway), CD11b/c (microglial activation), and BAX (apoptosis) in the amygdala and spinal cord of db/db mice. Our results showed the antihyperglycemic and antiallodynic effects of CoPP treatment as well as the potent antioxidant, antinociceptive, anti-inflammatory, and antiapoptotic properties of this HO-1 inducer in the CNS of type 2 diabetic mice. Treatment with CoPP also prevented the downregulation of several antioxidant proteins (Nrf2, SOD-1, and NQO1) and/or enhanced the protein levels of HO-1 and GSTM1 in the spinal cord and/or amygdala of db/db mice. These effects might be implicated in the antiallodynic actions of CoPP. Our findings revealed the modulatory effects of CoPP in the CNS of db/db mice and provide new prospects for novel type 2 diabetes-associated neuropathy therapies.

Fabry disease (FD) is a X-linked lysosomal storage disorder caused by deficient function of the alpha-galactosidase A (α-GalA) enzyme. α-GalA deficiency leads to multisystemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide (Gb3). A hallmark symptom of FD patients is neuropathic pain that appears in the early stage of the disease as a result of peripheral small fiber damage. Previous studies have shown that Acetyl-L-carnitine (ALC) has neuroprotective, neurotrophic, and analgesic activity in animal models of neuropathic pain. To study the action of ALC on neuropathic pain associated with FD, we treated α-GalA gene null mice (α-GalA(-/0)) with ALC for 30 days. In α-Gal KO mice ALC treatment induced acute and long-lasting analgesia, which persisted 1 month after drug withdrawal. This effect was antagonized by single administration of LY341495, an orthosteric antagonist of mGlu2/3 metabotropic glutamate receptors. We also found an up-regulation of mGlu2 receptors in cultured DRG neurons isolated from 30-day ALC treated α-GalA KO mice. However, the up-regulation of mGlu2 receptors was no longer present in DRG neurons isolated 30 days after the end of treatment. Taken together, these findings suggest that ALC induces analgesia in an animal model of FD by up-regulating mGlu2 receptors, and that analgesia is maintained by additional mechanisms after ALC withdrawal. ALC might represent a valuable pharmacological strategy to reduce pain in FD patients.

The search for safe and efficient chronic pain treatments is dampened by the lack of reliable models that faithfully reproduce current pharmacological treatments for chronic spontaneous pain in humans. Preclinical models often assess the antinociceptive efficacy of non-contingent pharmacological treatments evaluated in the short-term. Here, we provide a protocol of contingent operant self-medication in mice, which allows the estimation of spontaneous pain relief and drug abuse liability in models of persistent pain. This paradigm requires preliminary habituation and animal handling, followed by training of mice in operant conditioning boxes, to allow subsequent analgesic drug self-administration. After the initial acquisition of food-maintained operant behavior, a chronic pain sensitization is induced. Posterior intravenous jugular catheterization and coupling of operant conditioning boxes to perfusion pumps allow quantification of operant responding for intravenous drug self-administration. All mice show an initial operant drug self-administration behavior associated with the previous food-maintained operant training. This initial operant responding is extinguished after administration of ineffective treatments, but continues when the compounds have analgesic efficacy or intrinsic reinforcing properties. The identification of a significant drug self-administration selectively expressed in mice exposed to the chronic pain condition is indicative of analgesic drug effects, whereas persistent self-administration in control mice is indicative of abuse liability. The present protocol provides the behavioral and surgical procedures needed to assess spontaneous pain relief and potential for abuse of pharmacological treatments, through contingent analgesic self-medication in mice. Graphic abstract: Animals are subjected to a 5-day food self-administration protocol with a fixed ratio of reinforcement of 1 (FR1, 1 interaction with the active nose-poke causes the release of 1 reinforcer/infusion), to acquire the operant behavior. After this training, mice are subjected to the chronic pain or sham procedure, and four days later an intravenous (i.v.) catheterization is performed, to allow self-administration with the selected compound or its vehicle. Three days after the catheterization, animals start the drug/vehicle self-administration protocol at FR1. The patency of the catheter is evaluated with the thiopental test after the last self-administration session. Adapted from Bura (2018).