International Journal of Legal Medicine

, Volume 126, Issue 5, pp 807–814 | Cite as

Cannabinoid receptor type 2 is time-dependently expressed during skin wound healing in mice

  • Ji-Long Zheng
  • Tian-Shui Yu
  • Xiao-Na Li
  • Yan-Yan Fan
  • Wen-Xiang Ma
  • Yu Du
  • Rui Zhao
  • Da-Wei Guan
Original Article


Dynamic localization of CB2R and quantitative analysis of CB2R mRNA during skin wound healing in mice were performed. Co-localization of CB2R with F4/80 or α-SMA was detected by double-color immunofluorescence microscopy. A total of 110 male mice were divided into control, injury, and postmortem groups. Sixty-five mice were sacrificed, followed by sampling at 0.5 h–21 days post-injury. Five mice without incision were used as control. The other 40 mice that received incised wound were sacrificed at 5 days after injury. The samples were collected at 0 h–3 days postmortem. In the uninjured controls, CB2R immunoreactivity was detected in the epidermis, hair follicles, sebaceous glands, dermomuscular layer, and vascular smooth muscle. In the incision groups, polymorphonulcear cells, macrophages, and myofibroblasts showed positive staining for CB2R. Morphometrically, the average ratios of CB2R-positive cells were more than 50 % at 5 days post-wounding, whereas it was <50 % at the other posttraumatic intervals. The average ratios of CB2R-positive macrophages maximized at 3 days post-wounding, and the average ratios of CB2R-positive myofibroblasts peaked at 5 days post-wounding. The relative quantity of CB2R mRNA expression maximized at posttraumatic 5 days in comparison with control as detected by real-time PCR, with an average ratio of >4.10, which was also confirmed by Western blotting. There was no significant change for CB2R protein within 6 h postmortem and for mRNA within 3 h postmortem as compared with the control group. In conclusion, dynamic distribution and expression of CB2R suggest that CB2R is involved in modulating macrophages and myofibroblasts in response to inflammatory event and repair process in mouse skin wound healing, and CB2R is available as a marker for wound age determination.


Wound age determination Skin incision CB2R Macrophage Myofibroblast Real-time PCR 



This study was financially supported in part by a grant from research funds for the Doctoral Program funded by the Ministry of Education of China (200801590020) and a grant funded by the National Natural Science Foundation of China (30271347).

Supplementary material

414_2012_741_Fig6_ESM.jpg (8 kb)
Supplemental Fig. 1

Average ratio of CB2R-positive cells in each group. a p < 0.05 (vs control group); b p < 0.05 (vs preceding group) (JPEG 7 kb)

414_2012_741_MOESM1_ESM.tif (88 kb)
High-resolution image (TIFF 88 kb)
414_2012_741_Fig7_ESM.jpg (9 kb)
Supplemental Fig. 2

The average ratios of CB2R to GAPDH at different postmortem intervals. a p < 0.05 (vs 0 h group and preceding group) (JPEG 9 kb)

414_2012_741_MOESM2_ESM.tif (163 kb)
High-resolution image (TIFF 162 kb)
414_2012_741_Fig8_ESM.jpg (8 kb)
Supplemental Fig. 3

Relative intensity of CB2R to GAPDH. The data of 0 h represent the results obtained from the control group. All values are expressed as the means ± SD (n = 5). *p < 0.05 (vs control group); **p < 0.05 (vs control group and preceding group) (JPEG 8 kb)

414_2012_741_MOESM3_ESM.tif (86 kb)
High-resolution image (TIFF 85 kb)
414_2012_741_Fig9_ESM.jpg (8 kb)
Supplemental Fig. 4

Relative intensity of CB2R to GAPDH. All values are expressed as the means ± SD (n = 5). *p < 0.05 (vs control group and preceding group) (JPEG 7 kb)

414_2012_741_MOESM4_ESM.tif (103 kb)
High-resolution image (TIFF 103 kb)
414_2012_741_Fig10_ESM.jpg (8 kb)
Supplemental Fig. 5

Relative quantity of CB2R mRNA by real-time PCR at different posttraumatic intervals. The data of 0 h represent the results obtained from the control group. All values represent the means ± SD (n = 5). *p < 0.05 (vs control group); **p < 0.05 (vs control group and preceding group) (JPEG 8 kb)

414_2012_741_MOESM5_ESM.tif (146 kb)
High-resolution image (TIFF 145 kb)
414_2012_741_Fig11_ESM.jpg (7 kb)
Supplemental Fig. 6

Relative quantity of CB2R mRNA by real-time PCR at different postmortem intervals. All values represent the means ± SD (n = 5). *p < 0.05 (vs control group and preceding group) (JPEG 7 kb)

414_2012_741_MOESM6_ESM.tif (88 kb)
High-resolution image (TIFF 87 kb)


  1. 1.
    Munro S, Thomas KL, Abu-Shaar M (1993) Molecular characterization of a peripheral receptor for cannabinoids. Nature 365:61–65PubMedCrossRefGoogle Scholar
  2. 2.
    Kishimoto S, Muramatsu M, Gokoh M, Oka S, Waku K, Sugiura T (2005) Endogenous cannabinoid receptor ligand induces the migration of human natural killer cells. J Biochem 137:217–223PubMedCrossRefGoogle Scholar
  3. 3.
    Oka S, Ikeda S, Kishimoto S, Gokoh M, Yanagimoto S, Waku K, Sugiura T (2004) 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces the migration of EoL-1 human eosinophilic leukemia cells and human peripheral blood eosinophils. J Leukoc Biol 76:1002–1009PubMedCrossRefGoogle Scholar
  4. 4.
    Facci L, Dal Toso R, Romanello S, Buriani A, Skaper SD, Leon A (1995) Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide. Proc Natl Acad Sci U S A 92:3376–3380PubMedCrossRefGoogle Scholar
  5. 5.
    Matias I, Pochard P, Orlando P, Salzet M, Pestel J, Di Marzo V (2002) Presence and regulation of the endocannabinoid system in human dendritic cells. Eur J Biochem 269:3771–3778PubMedCrossRefGoogle Scholar
  6. 6.
    Galiègue S, Mary S, Marchand J, Dussossoy D, Carrière D, Carayon P, Bouaboula M, Shire D, Le Fur G, Casellas P (1995) Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur J Biochem 232:54–61PubMedCrossRefGoogle Scholar
  7. 7.
    Mackie K (2006) Cannabinoid receptors as therapeutic targets. Annu Rev Pharmacol Toxicol 46:101–122PubMedCrossRefGoogle Scholar
  8. 8.
    Buckley NE (2008) The peripheral cannabinoid receptor knockout mice: an update. Br J Pharmacol 153:309–318PubMedCrossRefGoogle Scholar
  9. 9.
    Brown AJ (2007) Novel cannabinoid receptors. Br J Pharmacol 152:567–575PubMedCrossRefGoogle Scholar
  10. 10.
    Ständer S, Schmelz M, Metze D, Luger T, Rukwied R (2005) Distribution of cannabinoid receptor 1 (CB1) and 2 (CB2) on sensory nerve fibers and adnexal structures in human skin. J Dermatol Sci 38:177–188PubMedCrossRefGoogle Scholar
  11. 11.
    Karsak M, Gaffal E, Date R, Wang-Eckhardt L, Rehnelt J, Petrosino S, Starowicz K, Steuder R, Schlicker E, Cravatt B, Mechoulam R, Buettner R, Werner S, Di Marzo V, Tüting T, Zimmer A (2007) Attenuation of allergic contact dermatitis through the endocannabinoid system. Science 316:1494–1497PubMedCrossRefGoogle Scholar
  12. 12.
    Oka S, Wakui J, Ikeda S, Yanagimoto S, Kishimoto S, Gokoh M, Nasui M, Sugiura T (2006) Involvement of the cannabinoid CB2 receptor and its endogenous ligand 2-arachidonoylglycerol in oxazolone-induced contact dermatitis in mice. J Immunol 177:8796–8805PubMedGoogle Scholar
  13. 13.
    Ueda Y, Miyagawa N, Matsui T, Kaya T, Iwamura H (2005) Involvement of cannabinoid CB(2) receptor-mediated response and efficacy of cannabinoid CB(2) receptor inverse agonist, JTE-907, in cutaneous inflammation in mice. Eur J Pharmacol 520:164–171PubMedCrossRefGoogle Scholar
  14. 14.
    Akhmetshina A, Dees C, Busch N, Beer J, Sarter K, Zwerina J, Zimmer A, Distler O, Schett G, Distler JH (2009) The cannabinoid receptor CB2 exerts antifibrotic effects in experimental dermal fibrosis. Arthritis Rheum 60:1129–1136PubMedCrossRefGoogle Scholar
  15. 15.
    Kondo T, Ohshima T (1996) The dynamics of inflammatory cytokines in the healing process of mouse skin wound: a preliminary study for possible wound age determination. Int J Legal Med 108:231–267PubMedCrossRefGoogle Scholar
  16. 16.
    Montecucco F, Burger F, Mach F, Steffens S (2008) CB2 cannabinoid receptor agonist JWH-015 modulates human monocyte migration through defined intracellular signaling pathways. Am J Physiol Heart Circ Physiol 294:H1145–H1155Google Scholar
  17. 17.
    Raborn ES, Marciano-Cabral F, Buckley NE, Martin BR, Cabral GA (2008) The cannabinoid delta-9-tetrahydrocannabinol mediates inhibition of macrophage chemotaxis to RANTES/CCL5: linkage to the CB2 receptor. J Neuroimmune Pharmacol 3:117–129PubMedCrossRefGoogle Scholar
  18. 18.
    Michalski CW, Maier M, Erkan M, Sauliunaite D, Bergmann F, Pacher P, Batkai S, Giese NA, Giese T, Friess H, Kleeff J (2008) Cannabinoids reduce markers of inflammation and fibrosis in pancreatic stellate cells. PLoS One 3:e1701PubMedCrossRefGoogle Scholar
  19. 19.
    Ortiz-Rey JA, Suárez-Peñaranda JM, Da Silva EA, Muñoz JI, San Miguel-Fraile P, De la Fuente-Buceta A, Concheiro-Carro L (2002) Immunohistochemical detection of fibronectin and tenascin in incised human skin injuries. Forensic Sci Int 126:118–122PubMedCrossRefGoogle Scholar
  20. 20.
    Liu N, Chen Y, Huang X (2006) Fibronectin EIIIA splicing variant: a useful contribution to forensic wounding interval estimation. Forensic Sci Int 162:178–182PubMedCrossRefGoogle Scholar
  21. 21.
    Dressler J, Bachmann L, Strejc P, Koch R, Müller E (2000) Expression of adhesion molecules in skin wounds: diagnostic value in legal medicine. Forensic Sci Int 113:173–176PubMedCrossRefGoogle Scholar
  22. 22.
    Grellner W, Vieler S, Madea B (2005) Transforming growth factors (TGF-alpha and TGF-beta1) in the determination of vitality and wound age: immunohistochemical study on human skin wounds. Forensic Sci Int 153:174–180PubMedCrossRefGoogle Scholar
  23. 23.
    Grellner W (2002) Time-dependent immunohistochemical detection of proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha) in human skin wounds. Forensic Sci Int 130:90–96Google Scholar
  24. 24.
    Takamiya M, Saigusa K, Kumagai R, Nakayashiki N, Aoki Y (2005) Studies on mRNA expression of tissue-type plasminogen activator in bruises for wound age estimation. Int J Legal Med 119:16–21PubMedCrossRefGoogle Scholar
  25. 25.
    Bai R, Wan L, Shi M (2008) The time-dependent expressions of IL-1beta, COX-2, MCP-1 mRNA in skin wounds of rabbits. Forensic Sci Int 175:193–197PubMedCrossRefGoogle Scholar
  26. 26.
    Bauer M (2007) RNA in forensic science. Forensic Sci Int Genet 1:69–74PubMedCrossRefGoogle Scholar
  27. 27.
    Kondo T, Ishida Y (2010) Molecular pathology of wound healing. Forensic Sci Int 203:93–98PubMedCrossRefGoogle Scholar
  28. 28.
    Cecchi R (2010) Estimating wound age: looking into the future. Int J Legal Med 124:523–536PubMedCrossRefGoogle Scholar
  29. 29.
    Yu TS, Cheng ZH, Li LQ, Zhao R, Fan YY, Du Y, Ma WX, Guan DW (2010) The cannabinoid receptor type 2 is time-dependently expressed during skeletal muscle wound healing in rats. Int J Legal Med 124:397–404PubMedCrossRefGoogle Scholar
  30. 30.
    Ma WX, Yu TS, Fan YY, Zhang ST, Ren P, Wang SB, Zhao R, Pi JB, Guan DW (2011) Time-dependent expression and distribution of monoacylglycerol lipase during the skin-incised wound healing in mice. Int J Legal Med 125:549–558PubMedCrossRefGoogle Scholar
  31. 31.
    Ohshima T, Sato Y (1998) Time-dependent expression of interleukin-10 (IL-10) mRNA during the early phase of skin wound healing as a possible indicator of wound vitality. Int J Legal Med 111:251–255PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Ji-Long Zheng
    • 1
    • 2
  • Tian-Shui Yu
    • 1
    • 3
  • Xiao-Na Li
    • 4
  • Yan-Yan Fan
    • 1
  • Wen-Xiang Ma
    • 1
  • Yu Du
    • 2
  • Rui Zhao
    • 1
  • Da-Wei Guan
    • 1
  1. 1.Department of Forensic PathologyChina Medical University School of Forensic MedicineShenyangPeople’s Republic of China
  2. 2.Department of Forensic MedicineChina Criminal Police UniversityShenyangPeople’s Republic of China
  3. 3.Key Laboratory of Evidence ScienceChina University of Political Science and Law, Ministry of EducationBeijingPeople’s Republic of China
  4. 4.Department of ChemistryChina Medical University School of Basic MedicineShenyangPeople’s Republic of China

Personalised recommendations