Complete Freund's adjuvant-induced decrement of pruriceptor-mediated suppression of itch.

Peripheral inflammation is always accompanied by a noxious sensation, either pain or itch, providing a protective warning for the occurrence of pathological changes; however, the mechanisms determining whether pain, itch, or both will be elicited under certain inflammatory statuses are still far from clear. Complete Freund's adjuvant (CFA) contains heat killed and dried Mycobacterium tuberculosis widely used to induce inflammatory pain models, but how CFA treatment affects itch sensation and the possible mechanisms are still unclear. In this study, using itch behavior testing and calcium imaging, we showed that both the behaviors and calcium responses associated with Transient Receptor Potential Vanilloid 1 (TRPV1)-mediated histamine-dependent itch and Transient Receptor Potential Ankyrin 1 (TRPA1)-mediated histamine-independent itch were significantly suppressed by CFA treatment. Furthermore, to explore the possible cellular mechanisms, high-throughput single-cell RNA sequencing and real-time PCR were used to detect CFA-induced changes of itch-related genes in dorsal root ganglion (DRG) neurons. Our results revealed that although both nociceptive Trpv1+ and Trpa1+ DRG neurons were increased after CFA treatment, most known pruriceptors, including Hrh1+, Mrgpra3+, Mrgprd+, Htr3a+, Htr1f+, IL31ra+, Osmr+, and Lpar3+ DRG neurons, were significantly decreased, which may explain that CFA treatment caused itch suppression. This study indicated that itch sensation was affected after CFA treatment, although negatively, and comprehensive but not specific suppression of different pruriceptors was observed after CFA treatment, suggesting that a unified adaptive change of increased pain and decreased itch will occur simultaneously under CFA-induced inflammatory conditions.

Spinal Serotonin 1A Receptor Contributes to the Analgesia of Acupoint Catgut Embedding by Inhibiting Phosphorylation of the N-Methyl-d-Aspartate Receptor GluN1 Subunit in Complete Freund's Adjuvant-Induced Inflammatory Pain in Rats.

Acupoint catgut embedding (ACE) is a widely used traditional Chinese medicine method to manage various diseases, including chronic inflammatory pain. We sought to assess the possible analgesic effects of ACE in comparison with electroacupuncture (EA) and to study the analgesic mechanisms of ACE in a rat model of inflammatory pain induced by injection of complete Freund's adjuvant (CFA) into the hind paw of rats. The von Frey, radiant heat, and gait analysis tests were performed to evaluate the analgesic effects of ACE and EA, and Western blot and immunohistochemistry assays were carried out to determine the molecular mechanisms of ACE. ACE treatments were administered every 4 days or every week with different acupoints (ipsilateral, contralateral, or bilateral ST36 and GB30 acupoints). The most effective ACE strategy for attenuating the nocifensive response induced by CFA injection was performing ACE once a week at ipsilateral ST36 in combination with GB30. EA treatment every other day at ipsilateral ST36 and GB30 showed comparable analgesic effects. ACE inhibited the increased activation of the GluN1 subunit of the N-methyl-d-aspartate receptor and the subsequent Ca2+-dependent signals (CaMKII, ERK, and CREB) that take place in response to CFA. The effects of ACE were similar to intrathecal injection of vilazodone (a serotonin 1A receptor [5-HT1AR] agonist) and were blocked by WAY-100635 (a 5-HT1AR antagonist). In summary, we show that ACE attenuates CFA-induced inflammatory pain in rats by activating spinal 5-HT1AR and by inhibiting the phosphorylation of GluN1, thus, inhibiting the activation of Ca2+-dependent signaling cascades. PERSPECTIVE: This article presents the novel evidence concerning the spinal 5-HT1AR activation-related molecular signaling of ACE analgesia in a rat model of CFA-induced inflammatory pain. This work may help clinicians to verify the effectiveness of ACE analgesia and to better understand the underlying mechanism.

Analysis of the temporal expression of chemokines and chemokine receptors during experimental granulomatous inflammation: role and expression of MIP-1alpha and MCP-1.

1. Chemokine expression and function was monitored in an experimental model of granulomatous tissue formation after injection of croton oil in complete Freund's adjuvant (CO/CFA) into mouse dorsal air-pouches up to 28 days. 2. In the first week, mast cell degranulation and leukocyte influx (mononuclear cell, MNC, and polymorphonuclear cell, PMN) were associated with CXCR2, KC and macrophage inflammatory protein (MIP)-2 mRNA expression, as determined by TaqMan reverse transcriptase-polymerase chain reaction. KC ( approximately 400 pg x mg protein(-1), n=12) and MIP-2 (approximately 800 pg x mg protein(-1), n=12) proteins peaked at day 7, together with myeloperoxidase (MPO) activity. Highest MIP-1alpha (>1 ng x mg protein(-1), n=12) levels were measured at day 3. 3. After day 7, a gradual increase in CCR2 and CCR5 mRNA, monocyte chemoattractant protein (MCP)-1 mRNA and protein expression was measured. MCP-1 protein peaked at day 21 (approximately 150 pg x mg protein(-1), n=12) and was predominantly expressed by mast cells. A gradual increase in N-acetyl-beta-D-glucosaminidase (NAG) activity (maximal at 28 days) was also measured. 4. An antiserum against MIP-1alpha did not modify the inflammatory response measured at day 7 (except for a 50% reduction in MIP-1alpha levels), but provoked a significant increase in MPO, NAG and MCP-1 levels as measured at day 21 (n=6, P<0.05). An antiserum to MCP-1 reduced NAG activity at day 21 but increased MPO activity values (n=8, P<0.05). 5. In conclusion, we have shown that CO/CFA initiates a complex inflammatory reaction in which initial expression of MIP-1alpha serves a protective role whereas delayed expression of MCP-1 seems to have a genuine pro-inflammatory role.