Fig 1: Changes in CB1R in the vlPAG and dlPAG after i.n. administration of MCH in the CFA-induced inflammatory pain model. Histological examination of immunofluorescent tissue sections from the vlPAG (A–I) and dlPAG (J–R) of mice showing the activation of CB1R in the vlPAG and dlPAG 2 h after the i.n. administration of MCH (10 µg/30 µl) on the 4th day after CFA injection. A MCH1R antagonist, TC-MCH 7c (10 mg/kg, i.p.), was administered 30 min prior to treatment with MCH. Cells positive for CB1R (red) and/or c-Fos (green) in the vlPAG (A) and dlPAG (J) were visualized under a microscope. CB1R- and/or c-Fos-positive cells in the vlPAG (B–D) and dlPAG (K–M) were counted. PAG structure: vlPAG region (G) and dlPAG region (P). Scale bar: 100 and 30 µm. All groups: n = 6. *p < 0.05, ***p < 0.001 vs. the Control group, ### p < 0.001 vs. the CFA group, and &&& p < 0.001 vs. MCH group. The data were analyzed with a one-way ANOVA followed by Newman–Keuls post hoc tests (B–D and G–I). Data are expressed as means ± SEM. The expression levels and activation of CB1R in the vlPAG and dlPAG, respectively, were correlated with paw withdrawal frequency (E,F,N, and O) or with those in the mPFC (H,I,Q, and R). The r-values were analyzed with the Spearman rank correlation coefficient. vlPAG: ventrolateral PAG; dlPAG: dorsolateral PAG; CB1R: cannabinoid 1 receptor.
Fig 2: Immunohistochemical expression in human testis tissue of the two canonical EC receptors; CNR1 and CNR2. The images on the left show a general expression pattern, and a higher magnification of an area within a stippled line is shown on the right. All pictures show a low magnification image of negative controls in the right upper corner. CNR1 staining (upper images) is visible within nuclei of primary spermatocytes and in the cytoplasm of Leydig cells. CNR2 (bottom images) is localised in the cytoplasm of all types of germ cells, except early spermatocytes, and in somatic cells, especially in Leydig cells and peritubular cells. L (leptotene) and P (pachytene) indicate early and late spermatocytes, respectively.
Fig 3: Effects of opioid and cannabinoid receptor antagonists on the analgesic effects of i.n. MCH following CFA-induced inflammatory pain and PSNL-evoked neuropathic pain. Experimental design of CFA (A) and PSNL (B). Opioid (naltrexone, 5 mg/kg, i.p.) and CB1R (AM251, 4 mg/kg, i.p.) antagonist treatments (C,E, and G) and a1 adrenergic (prazosin, 1 mg/kg; i.p), a2 adrenergic (yohimbine, 1 mg/kg, i.p.), and 5-HT1/2 (cyproheptadine, 1 mg/kg, i.p.) receptor antagonists (D,F, and H) were administered 30 min prior to treatment with either MCH (10 µg/30 µl, i.n.) or saline. The behavioral tests were performed 30 and 60 min after MCH or saline treatment in the CFA-induced inflammatory pain model (C,D,E, and F; n = 6–7/group). In the PSNL-evoked neuropathic pain model, the behavioral tests were performed 30 min after saline or MCH (10 µg/30 µl/day for 7 days, i.n.) treatment (G,H; n = 6/group). The von Frey test (C,D,G, and H) and hot-plate test (E,F). ***p < 0.001 vs. the CFA or PSNL groups. # p < 0.05, ## p < 0.01, ### p < 0.001 compared with the MCH (10 µg/30 µl) group. All data were analyzed with a two-way RMANOVA followed by post hoc Bonferroni tests. The results are expressed as means ± SEM. i.n.: intranasal, i.p.: intraperitoneal.
Fig 4: Expression profiles of transcript isoforms coding for CB1, CB1A, CB1B and CB2A. Quantitative PCR (qPCR) experiments were performed on cDNA from isolated human Leydig cells (n = 4; LC), peritubular cells (n = 2; PC), Sertoli cells (n = 2; SC), pachytene spermatocytes (n = 4; SPC), round spermatids (n = 4; SPT) and total testis tissue (n = 4; TT). Histograms represent mean expression (relative to RPLP0) ± SEM. Following evaluation of normality (Shapiro–Wilk test and D’Agostino’s K-squared test) and of homogeneity of variances (Brown-Forsythe test), global analysis of variances was achieved with One-way ANOVA (CB1 and CB1A) or with Kruskal–Wallis one-way analysis of variance (CB1B and CB2A). Pairwise comparisons were also performed using unpaired Student’s t-test (CB1 and CB1A) or with Mann–Whitney U test (CB1B and CB2A). One, two or three stars denote statistical significance with p < 0.05, p < 0,01 or p < 0.005, respectively. p-values close to significance are also indicated.
Fig 5: Changes in CB1R in the mPFC after i.n. administration of MCH in the CFA-induced inflammatory pain model. Representative figure showing the mPFC region in the mouse brain (A) and the distribution of MCH in the mPFC 30 min after the i.n. administration of MCH-FITC (10 µg/30 µl) or saline. DAPI: blue; MCH-FITC: green. Scale bar: 400 µm and 30 µm (B). Representative graphs showing the expression levels and activation of CB1R in the mPFC 2 h after the i.n. administration of MCH (10 µg/30 µl) on the 4th day after CFA injection. A MCH1R antagonist, TC-MCH 7c (10 mg/kg, i.p.), was administered 30 min prior to treatment with MCH. CB1R- and/or c-Fos-positive cells in the mPFC were counted. Scale bar: 100 and 30 µm (C). The results were analyzed with a one-way ANOVA followed by Newman–Keuls post hoc tests (D–F). n = 6/group. ***p < 0.001 vs. the Control group, ### p < 0.001 vs. the CFA group, and &&& p < 0.001 vs. MCH group. Data are expressed as means ± SEM. Figures show data for the correlation between mechanical allodynia and a change in CB1R after i.n. MCH administration. The r-values were analyzed using a Spearman rank correlation coefficient. mPFC: medial prefrontal cortex; CB1R: cannabinoid 1 receptor; MCH1R: MCH 1 receptor.
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