The Changes Of Cannabinoid Type 2 Receptor Expression In Relation To Wound Age After Brain Contusion In Mice

Objective:The cannabinoid type 2 receptor(CB2R)belongs to the endogenous cannabinoid system and is a seven-transmembrane G protein-coupled receptor.Previous studies by our group have shown that the expression of CB2R in the healing process of skin and skeletal muscle injury is time-dependently,which may be as a marker for skin and skeletal muscle wound age estimation.Recent studies have shown that CB2R expression in the central nervous system is low under normal conditions but it is highly inducible.Under some pathological conditions such as brain contusion,the expression of CB2R can change rapidly,which can regulate inflammatory response,and exerts neuroprotective effects.The above findings suggest that the expression of CB2R after brain contusion may be associated with injury time.In this study,the expression changes of CB2R in cortex was examined at different time points after brain contusion in mice,and explored whether it can be used as a biological indicator for brain wound age eeatimation.Methods:72 Healthy clean male C57BL/6 mice,8 weeks old,each weighing 23-28g were randomly divided into the sham group and the 0.5h,1h,3h,6h,12h,1d,3d,7d,14d,21d and 28d groups post-contusin,with 6 mices in each group A brain contusion model was established by PCI3000 device.In the sham group,only the skull bone window was established.After intraperitoneal anesthesia,the mice heart should be fully exposed.The phosphate buffered saline(PBS)about 50ml was slowly instilled through the left ventricle.The whole brain was cut in half along the middle of the lesion after collected,half of the brain was used to extracted 2mm cortex tissue around the lesion,which was used for Western blotting assay,and the other half brian was fixed with 4%paraformaldehyde/PBS for histological examination.The expression of CB2R and its expression in different cell types were observed by immunohistochemistry and immunofluorescence staining.Results:1.HE staining results showed that the tissue hemorrhage,edema and cell necrosis gradually increased in the early stage after brain contusion.Inflammatory cell infiltration increased gradually after injury,and the necrotic tissue liquefaction was absorbed along with neovascularization and glial cell proliferation in the middle and late stage after brain contusion.2.Immunohistochemical staining results showed that only some of the neurons were positive for CB2R in the sham cortex.At 0.5h-12h,The ratio of CB2R~+cells increased gradually and reached the peak at the first time at 12h after injury,then decreased gradually until 3d post-brain contusion.The ratio of CB2R~+cells reached the peak again at 7d pos-injury.Then,it gradually decreased again and returned to the normal level at 28d after injury.3.Western blot results were consistent with immunohistochemical staining results.4.Immunofluorescent staining demonstrated that CB2R was expressed in neurons,monocytes and astrocytes after brain contusion.The changes of CB2R~+NeuN~+,CB2R~+CD68~+,CB2R~+GFAP~+cells ratio showed a single peak pattern,which peaked at 12h,1d and 7d post-injury,respectively.Conclusion:The expression of CB2R in neurons?monocytes and astrocytes after brain contusion in mice suggests that it may be involved in the regulation of the biological functions of those cells,and there is a time-dependent expression of CB2R as a whole or in single cell,suggesting its potential applicability as a biomarker to wound age estimation for brain contusion in forensic practice.