Elsevier

Neuroscience

Volume 135, Issue 1, 2005, Pages 235-245
Neuroscience

Pain mechanism
Peripheral nerve injury induces cannabinoid receptor 2 protein expression in rat sensory neurons

https://doi.org/10.1016/j.neuroscience.2005.06.009Get rights and content

Abstract

We have localized cannabinoid receptor 2 protein in rat and mouse somatic sensory nervous system, using an antibody that recognizes mouse cannabinoid receptor 2. Little or no cannabinoid receptor 2 immunoreactivity was found in sections of naive rat or mouse dorsal root ganglia or spinal cord. This was in accord with the lack of detectable cannabinoid receptor 2 mRNA in (dorsal root ganglion) neurons by in situ hybridization experiments described in the literature. However, we could detect cannabinoid receptor 2 immunoreactivity following unilateral nerve damage—either by sciatic nerve section, or by spinal nerve ligation. It was localized to the superficial laminae of the dorsal horn of the spinal cord, ipsilateral to the nerve damage, coincident with the area of termination of damaged afferents which was marked by loss of isolectin B4 binding. This upregulation was not seen in cannabinoid receptor 2 null mice. The cannabinoid receptor 2 protein in spinal cord appeared to be expressed on sensory neuron afferent terminals as it colocalized with two markers of damaged afferents, namely growth associated protein-43 and the neuropeptide galanin. Moreover, it did not colocalize with markers of activated microglial cells (OX-42) or astroglial cells (glial fibrillary acidic protein) in rat spinal cord. In the peripheral nerve, accumulation of cannabinoid receptor 2 immunoreactivity was seen in nerve sections proximal, but not distal, to the ligation site, suggesting transport down the nerve from the cell bodies. Although convincing cannabinoid receptor 2 immunoreactivity was seen in neither uninjured nor injured dorsal root ganglion neuron cell bodies in tissue sections, expression was detectable in isolated, cultured neurons that had received a prior axotomy in vivo.

This clear demonstration of CB2 receptors on sensory neurons suggests an additional cellular target for CB2 agonist induced analgesia, at least in neuropathic models.

Section snippets

Animals and surgical methods

Adult male Wistar rats (120–140g; Charles River UK Ltd, Kent, UK), were used in this study. Three animals were used in each group for histochemical analysis. All surgery was performed under enflurane/O2 inhalation anesthesia, according to UK Home Office-approved procedures. All experiments were performed according to Home Office (United Kingdom) guidelines and with approval of the local Novartis Animal Welfare and Ethics committee. The number of animals used and their suffering was minimised.

Results

Anti-CB2 antibody was first tested on cultured control CHO cells and CHO cells expressing CB2 receptor, as well as sections of rat spleen. Fig. 1 shows validation of the CB2 directed antibody by immunolabeling on CB2 expressing (A) but not control CHO cells (B) in culture. Clear staining of CB2 expressing cells is seen in spleen sections as previously described Fig. 1C.

There was no convincing staining in naïve spinal cord or DRG (not shown). However, 14 days following L5 spinal nerve ligation

Discussion

CB2 agonists have been receiving much attention recently as activators of the cannabinoid system that have analgesic effects without the undesirable, centrally mediated, side effects of brain penetrate CB1 ligands. In preclinical studies the CB2 selective agonist HU-308 reduced the paw licking response following formalin injection (Hanus et al., 1999) and the reported CB2 selective agonist AM1241 inhibited both mechanical and thermal hypersensitivity in animals with spinal nerve ligation

Acknowledgments

We would like to thank Natalie Stoakely, Clive Gentry and Sadhana Patel for carrying out the surgeries.

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