A new CB₂R transgenic mouse model for the study of inflammation

N. Aparicio¹, M. T. Grande¹, A. Lopez-Vivo², I. Bermejo¹, M. A. Barreda-Manso¹, R. Tolon¹, C. Hillard³, J. Romero¹. ¹Francisco de Vitoria University, Pozuelo de Alarcon, Madrid, Spain, ²Hospital Universitario Fundacion Alcorcon, Alcorcon, Madrid, Spain, ³Medical College of Wisconsin, Milwaukee, WI.

Introduction: Because of limited availability of research tools, cannabinoid CB₂ receptors (CB₂R) have so far been difficult to study. To solve this problem, we generated a new transgenic mouse model, which expresses the reporter gene (Green Fluorescent Protein, GFP) under the control of the endogenous mouse Cb2 promoter.CB₂R are expressed abundantly in cells of the immune system, including immunocompetent cells of the CNS, such as microglia. Thus, in the present study we analyzed the expression of CB₂R in two models of inflammation, one affecting the CNS (Alzheimer’s disease) and the second related with liver fibrosis pathology.

Method:

CB₂R mouse model: generated by inserting an EGFP reporter gene preceded by an IRES sequence in the 3’ UTR of the Cb2 mouse gene. This approach results in the expression of the reporter gene under the control of the endogenous mouse Cb2 promoter in a bicistronic mRNA with the CB₂ protein.

CB₂R mouse model characterization in inflammation

Alzheimer’s disease: CB₂-GFP transgenic mice were crossed with 5xFAD mice. Immunohistochemical stainings were performed against EGFP in order to describe CB₂R expression in the β-amyloid (Aβ) pathology. In addition, primary cultures of microglial cells were treated with Aβ to study changes in EGFP-expression.

Bile duct ligation: Adult CB₂-GFP transgenic mice underwent bile duct ligation and sham-operated animals were used as controls. Organ histology and immunohistochemical staining were performed to assess the expression of inflammatory and fibrotic markers in the livers from CB₂GFP mice.

Results:

Alzheimer’s disease: EGFP flourescence was increased in in vitro microglial cells treated with Aβ. EGFP was detected in microglial cells associated to neuritic plaques under chronic neuroinflammation conditions. No EGFP was evident in brain regions lacking Aβ plaques.

Liver fibrosis: Bile-duct ligation caused profound hepatic changes as showed by increases in serum AST, ALT, and total and direct bilirrubin levels. EGFP expression was induced in endothelial cells and inflammatory cells in damaged livers.

Conclusion: This new CB₂R mouse model allows for a better understanding of the expression and role of CB₂R in different models of inflammation, offering a powerful tool for the development of new therapeutic drugs.

References:

- Benito C et al, J. Neurosci 23, 11136-11141, 2003.

- Mahmoud MF et al, Eur. J. Pharmacol. 742, 118-124, 2014.

- Mallat A et al, J. Hepatol. 59, 891-896, 2013.