The cannabinoid receptor type 2 is time-dependently expressed during skeletal muscle wound healing in rats

Abstract

The expression of the cannabinoid receptor type 2 (CB2R) was investigated by immunohistochemistry, Western blotting, and RT-PCR during wound healing of contused skeletal muscle in rats with attempt of its applicability to skeletal muscle wound age estimation. Furthermore, Macrophage Marker (MAC387) was utilized to identify macrophages recruited into injured skeletal muscle tissue. Co-localization of CB2R with Macrophage Marker was detected by confocal laser scanning microscopy. A total of 50 Sprague–Dawley male rats were divided into control and contusion groups (3 h, 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury). In the uninjured controls, immunoreactivity of CB2R was detected in the sarcolemma and sarcoplasm of normal myofibers. In the contusion groups, a few polymorphonulcear cells, a large number of macrophages, and spindle-shaped fibroblastic cells showed a positive staining for CB2R in wounded zones. By Western blotting analysis, the average of CB2R to GAPDH ratios in 5–7 days post-injury groups was highest, and all the samples had ratios of >2.60. In the other groups, no samples showed ratios of >2.60 and the CB2R to GAPDH ratios ranged from 1.19 to 2.59. The expression tendency was also confirmed by RT-PCR. From the viewpoint of forensic pathology, these observations suggested that the ratio markedly exceeding 2.60 strongly indicated a wound age of 5–7 days. In conclusion, dynamic distribution and expression of CB2R suggest that CB2R be involved in modulating macrophages in response to inflammatory event in rat skeletal muscle wound healing and CB2R be available as a marker for wound age determination.

This is a preview of subscription content, access via your institution.

We’re sorry, something doesn't seem to be working properly.

Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. 1.

    Munro S, Thomas KL, Abu-Shaar M (1993) Molecular characterization of a peripheral receptor for cannabinoids. Nature 365:61–65

    CAS  Article  PubMed  Google 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 137:217–223

    CAS  Article  PubMed  Google 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 76:1002–1009

    CAS  Article  PubMed  Google 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 92:3376–3380

    CAS  Article  PubMed  Google 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 269:3771–3778

    CAS  Article  PubMed  Google 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 232:54–61

    Article  PubMed  Google Scholar 

  7. 7.

    Mackie K (2006) Cannabinoid receptors as therapeutic targets. Annu Rev Pharmacol Toxicol 46:101–122

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Buckley NE (2008) The peripheral cannabinoid receptor knockout mice: an update. Br J Pharmacol 153:309–318

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Brown AJ (2007) Novel cannabinoid receptors. Br J Pharmacol 152:567–575

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Wright K, Rooney N, Feeney M, Tate J, Robertson D, Welham M, Ward S (2005) Differential expression of cannabinoid receptors in the human colon: cannabinoids promote epithelial wound healing. Gastroenterology 129:437–453

    PubMed  Google Scholar 

  11. 11.

    Montecucco F, Lenglet S, Braunersreuther V, Burger F, Pelli G, Bertolotto M, Mach F, Steffens S (2009) CB(2) cannabinoid receptor activation is cardioprotective in a mouse model of ischemia/reperfusion. J Mol Cell Cardiol 46:612–620

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Bátkai S, Osei-Hyiaman D, Pan H, El-Assal O, Rajesh M, Mukhopadhyay P, Hong F, Harvey-White J, Jafri A, Haskó G, Huffman JW, Gao B, Kunos G, Pacher P (2007) Cannabinoid-2 receptor mediates protection against hepatic ischemia/reperfusion injury. FASEB J 21:1788–1800

    Article  PubMed  Google Scholar 

  13. 13.

    Ashton JC, Rahman RM, Nair SM, Sutherland BA, Glass M, Appleton I (2007) Cerebral hypoxia–ischemia and middle cerebral artery occlusion induce expression of the cannabinoid CB2 receptor in the brain. Neurosci Lett 412:114–117

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Muñoz-Luque J, Ros J, Fernández-Varo G, Tugues S, Morales-Ruiz M, Alvarez CE, Friedman SL, Arroyo V, Jiménez W (2008) Regression of fibrosis after chronic stimulation of cannabinoid CB2 receptor in cirrhotic rats. J Pharmacol Exp Ther 324:475–483

    Article  PubMed  Google Scholar 

  15. 15.

    Cavuoto P, McAinch AJ, Hatzinikolas G, Janovská A, Game P, Wittert GA (2007) The expression of receptors for endocannabinoids in human and rodent skeletal muscle. Biochem Biophys Res Commun 364:105–110

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Lighthall JW (1988) Controlled cortical impact: a new experimental brain injury model. J Neurotrauma 5:1–15

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    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 119:16–21

    Article  PubMed  Google Scholar 

  18. 18.

    Kondo T (2007) Timing of skin wounds. Leg Med 9:109–114

    Article  Google Scholar 

  19. 19.

    Takamiya M, Fujita S, Saigusa K, Aoki Y (2008) Simultaneous detection of eight cytokines in human dermal wounds with a multiplex bead-based immunoassay for wound age estimation. Int J Legal Med 122:143–148

    Article  PubMed  Google Scholar 

  20. 20.

    Ishida Y, Kimura A, Takayasu T, Eisenmenger W, Kondo T (2008) Expression of oxygen-regulated protein 150 (ORP150) in skin wound healing and its application for wound age determination. Int J Legal Med 122:409–414

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Guan DW, Ohshima T, Kondo T (2000) Immunohistochemical study on Fas and Fas ligand in skin wound healing. Histochem J 32:85–91

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Kondo T, Ohshima T, Mori R, Guan DW, Ohshima K, Eisenmenger W (2002) Immunohistochemical detection of chemokines in human skin wounds and its application to wound age determination. Int J Legal Med 116:87–91

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Takamiya M, Saigusa K, Nakayashiki N, Aoki Y (2003) Studies on mRNA expression of basic fibroblast growth factor in wound healing for wound age determination. Int J Legal Med 117:46–50

    PubMed  Google Scholar 

  24. 24.

    Hayashi T, Ishida Y, Kimura A, Takayasu T, Eisenmenger W, Kondo T (2004) Forensic application of VEGF expression to skin wound age determination. Int J Legal Med 118:320–325

    Article  PubMed  Google Scholar 

  25. 25.

    Kagawa S, Matsuo A, Yagi Y, Ikematsu K, Tsuda R, Nakasono I (2009) The time-course analysis of gene expression during wound healing in mouse skin. Leg Med 11:70–75

    CAS  Article  Google Scholar 

  26. 26.

    Zhao R, Guan DW, Zhang W, Du Y, Xiong CY, Zhu BL, Zhang JJ (2009) Increased expressions and activations of apoptosis-related factors in cell signaling during incised skin wound healing in mice: a preliminary study for forensic wound age estimation. Leg Med 11:S155–S160

    Article  Google Scholar 

  27. 27.

    McLennan IS (1996) Degenerating and regenerating skeletal muscles contain several subpopulations of macrophages with distinct spatial and temporal distributions. J Anat 188:17–28

    PubMed  Google Scholar 

  28. 28.

    Pimorady-Esfahani A, Grounds MD, McMenamin PG (1997) Macrophages and dendritic cells in normal and regenerating murine skeletal muscle. Muscle Nerve 20:158–166

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Papadimitriou JM, Robertson TA, Mitchell CA, Grounds MD (1990) The process of new plasmalemma formation in focally injured skeletal muscle fibres. J Struct Biol 103:124–134

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Robertson TA, Maley MA, Grounds MD, Papadimitriou JM (1993) The role of macrophages in skeletal muscle regeneration with particular reference to chemotaxis. Exp Cell Res 207:321–331

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Chazaud B, Brigitte M, Yacoub-Youssef H, Arnold L, Gherardi R, Sonnet C, Lafuste P, Chretien F (2009) Dual and beneficial roles of macrophages during skeletal muscle regeneration. Exerc Sport Sci Rev 37:18–22

    Article  PubMed  Google Scholar 

  32. 32.

    Chazaud B, Sonnet C, Lafuste P, Bassez G, Rimaniol AC, Poron F, Authier FJ, Dreyfus PA, Gherardi RK (2003) Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth. J Cell Biol 163:1133–1143

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    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–H1155

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    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 3:117–129

    Article  PubMed  Google Scholar 

  35. 35.

    Sun JH, Wang YY, Zhang L, Gao CR, Zhang LZ, Guo Z (2010) Time-dependent expression of skeletal muscle troponin I mRNA in the contused skeletal muscle of rats: a possible marker for wound age estimation. Int J Legal Med 124:27–33

    Article  PubMed  Google Scholar 

  36. 36.

    Hurme T, Kalimo H, Lehto M, Järvinen M (1991) Healing of skeletal muscle injury: an ultrastructural and immunohistochemical study. Med Sci Sports Exerc 23:801–810

    CAS  PubMed  Google Scholar 

  37. 37.

    Huard J, Li Y, Fu FH (2002) Muscle injuries and repair: current trends in research. J Bone Joint Surg Am 84:822–832

    PubMed  Google Scholar 

  38. 38.

    Wright-Carpenter T, Opolon P, Appell HJ, Meijer H, Wehling P, Mir LM (2004) Treatment of muscle injuries by local administration of autologous conditioned serum: animal experiments using a muscle contusion model. Int J Sports Med 25:582–587

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    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 175:193–197

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgment

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

Author information

Affiliations

Authors

Corresponding author

Correspondence to Da-Wei Guan.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Yu, TS., Cheng, ZH., Li, LQ. et al. The cannabinoid receptor type 2 is time-dependently expressed during skeletal muscle wound healing in rats. Int J Legal Med 124, 397–404 (2010). https://doi.org/10.1007/s00414-010-0465-1

Download citation

Keywords

  • Forensic pathology
  • Wound age determination
  • Skeletal muscle contusion
  • CB2R
  • Macrophage