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Fig. 6 Blocking the peripheral activity of CB1 increases bone calcification and mineralization while decreasing osteoclast activity. Zebrafish were exposed to either AM6545 (B) or JD5037 (C) from 48 to 72 hpf. Alazarin red staining was performed at 128 hpf to determine if there were any changes in calcification at the opercle bone, marked by the black arrows, after exposure (B,C) as compared to control (A). Both AM6545 (B) and JD5037 (C) showed a calcification increase as compared to the control (A) at 128 hpf. n = 12 for each treatment group. For AM6545 (B), 100% of zebrafish showed a significant increase in calcification as compared to the control (A). For JD5037 (C), 100% of zebrafish showed a significant increase in calcification as compared to both the control (A) and AM6545 (B). Zebrafish were exposed to AM6545 (E) and JD5037 (F) from 36 to 72 hpf and fixed at 144 hpf. At 144 hpf, Von Kossa staining was performed in order to observe any changes in mineralization after exposure (E,F) as compared to the control (D). Images were taken at 8x and 20x (D,E,F). Both AM6545 (E) and JD5037 (F) exposure led to a mineralization increase at 144 hpf as compared to the control (D) at 80x and 200x magnification. n = 10 for each treatment group. 100% of AM6545 (E) exposed zebrafish showed a significant increase mineralization as compared to the control (D). As for JD5037 (F), 100% of zebrafish showed a significant mineralization increase as compared to the control (D), and the AM6545 (E) exposed zebrafish. Tartrate Resistant Acid Phosphatase (T.R.A.P.) stain shows presence of osteoclasts. The zebrafish were exposed to AM6545 (H) and JD5037 (I) from 12 to 14 dpf. At 14 dpf, the larvae were fixed and TRAP was performed to determine any changes in osteoclast presence (G,H,I). Both AM6545 (H) and JD5037 (I) exposure showed a significant decrease in osteoclast presence at 14 dpf as compared to the control (G) at 14 dpf. n = 10 for each treatment group. For the control group (G) 100% showed high osteoclast activity that is common at 14 dpf. For AM6545 (H), 70% showed little to no osteoclast activity, and 30% showed a significant decrease in osteoclast presence as compared to the control. For JD5037 (I), 80% showed little to no osteoclast presence, and 20% showed a significant decrease in osteoclast presence. Overall, after exposure to either compound (H,I), 100% of the zebrafish showed a significant decrease in osteoclast presence as compared to the control (G).
Reprinted from Molecular and Cellular Endocrinology, 529, Fraher, D., Mann, R.J., Dubuisson, M.J., Ellis, M.K., Yu, T., Walder, K., Ward, A.C., Winkler, C., Gibert, Y., The endocannabinoid system and retinoic acid signaling combine to influence bone growth, 111267, Copyright (2021) with permission from Elsevier. Full text @ Mol. Cell. Endocrinol.