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The role of anandamide-synthesising pathways of primary sensory neurons in the development of pathological pain

Final Report Summary - ANANDAMIDE AND PAIN (The role of anandamide-synthesising pathways of primary sensory neurons in the development of pathological pain)


The endovanilloid/endocannabinoid systems control the activity and excitability of a major sub-population of nociceptive primary sensory neurons (PSN), through the signalling molecule anandamide (AEA) and its main targets, the excitatory transient receptor potential vanilloid type 1 ion channel (TRPV1) and the inhibitory cannabinoid 1 (CB1) receptor. AEA is thought to be synthesized by multiple enzymatic pathways. At present, however, the enzymatic pathway(s) involved in AEA synthesis in PSN is not known.


Here, using a multidisciplinary approach, we aimed not only to find the expression pattern of enzymes, which might take part in AEA production in PSN but also to determine their possible roles, if any, in the development of pain in inflammation and nerve injury origin.


RT-PCR analysis revealed that all the enzymes implicated in AEA synthesis are present in naive DRG, however, on sections only four (NAPE-PLD, Inpp5, GDE-1 and PTPn22) were detectable by conventional immunofluorescent staining. Using double immunohistochemical labeling, we found that NAPE-PLD and Inpp5 positive neurons are exclusively small in size and predominantly colabeled with isolectin B4 (markers of unmyelinated C-fiber neurons). More than half of NAPE-PLD labeled cells and approximately 80% of Inpp5 positive neurons showed TRPV1-immunoreactivity (marker of nociceptive DRG neurons). While PTPn22 is mainly expressed in the NF200-labeled subpopulation of medium- and large-sized DRG neurons, most of the GDE-1 immunreactive (-IR) cells costained with CGRP. GDE-1 and PTPn22 appear to be expressed by different population of neurons showing just low level of co-localization.

Expression profiles of these enzymes were altered on the effect of culturing as determined by quantitative real-time PCR and western immunoblotting.

Cell-size analyses of DRG soma derived from the ipsi- and contralateral sides of CFA-treated rats 3 day after injection and naive rats confirmed rightward shifts in the size of NAPE-PLD-, GDE-1- and TRPV1-IR neurons on both sides of inflamed tissue compared with those in naive rats. A leftward shift in the size distribution of PTPn22-IR neurons on both sides of CFA-injection was also observed.

Western immunoblottting's results revealed that after unilateral axotomy of L5 spinal nerve, all of the detectable enzymes (NAPE-PLD, GDE-1 and PTPn22) participating in AEA production as well as TRPV1 protein levels were significantly reduced in DRG exposed to ligation. Interestingly, at the same time, apparent compensatory change in the expression of TRPV1 receptor was also detected in the contralateral side of nerve injury. Based on cell-size analyses of DRGs of SNL- and sham-treated rats we found rightward shifts in the size of NAPE-PLD- and TRPV1-IR neurons in the ipsilateral ligated DRG as compared with the corresponding sham-operated controls. There was a significant leftward shift in PTPn22 immunreactivity after axotomy in both ipsilateral DRGs examined (ligated and non-ligated) in comparison with DRGs from sham-treated animals.


Our data indicate that several putative AEA-synthesising pathways could indeed participate in AEA production in PSN. Besides TRPV1 receptor, NAPE-PLD, GDE-1 and PTPn22 seem to be affected by inflammation of peripheral tissues and injury to peripheral nerves, at least, in DRG neurons. Unlike acute peripheral inflammation, nerve injury might cause, in general, some damage in the expression of proteins examined in DRG exposed to spinal nerve ligation. Our results also suggest that phenotypes of sensory neurons underwent profound changes at single cell level in response to these peripheral pain conditions, however, in case of inflammation, these alterations have been masked by the net effect of changes induced by CFA, and thus, certain proteins/enzymes seemingly may stay unaffected.

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