AUTHOR=Ducza László , Gajtkó Andrea , Hegedűs Krisztina , Bakk Erzsébet , Kis Gréta , Gaál Botond , Takács Roland , Szücs Péter , Matesz Klára , Holló Krisztina 

TITLE=Neuronal P2X4 receptor may contribute to peripheral inflammatory pain in rat spinal dorsal horn

JOURNAL=Frontiers in Molecular Neuroscience

VOLUME=Volume 16 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/molecular-neuroscience/articles/10.3389/fnmol.2023.1115685

DOI=10.3389/fnmol.2023.1115685

ISSN=1662-5099

ABSTRACT=Abstract
Intense inflammation may result in pain, which manifests as spinal central sensitisation. There is growing evidence that purinergic signaling plays a pivotal role in the orchestration of pain processing. Over the last decade the ionotropic P2X purino receptor 4 (P2X4) got into spotlight in neuropathic disorders, however its precise spinal expression was scantily characterised during inflammatory pain. Thus, we intended to analyse the receptor distribution within spinal dorsal horn and lumbar dorsal root ganglia (DRG) of rats suffering in inflammatory pain induced by complete Freund adjuvant (CFA). 
Methods
CFA- induced peripheral inflammation was validated by mechanical and thermal behavioural tests. In order to ensure about the putative alteration of spinal P2X4 receptor gene expression qPCR reactions were designed, followed by immunoperoxidase and Western blot experiments to assess changes at a protein level. Colocalisation of P2X4 with neuronal and glial markers was investigated by double immunofluorescent labelings, which were subsequently analysed with IMARIS software. Transmission electronmicroscopy was applied to study the ultrastructural localisation of the receptor. Concurrently, in lumbar DRG cells similar methodology has been carried out to complete our observations.
Results			
The figures of mechanical and thermal behavioural tests proved the establishment of CFA- induced inflammatory pain. We observed significant enhancement of P2X4 transcript level within the spinal dorsal horn three days upon CFA administration. Elevation of P2X4 immunoreactivity within Rexed lamina I-II of the spinal gray matter was synchronous with mRNA expression, and confirmed by protein blotting. According to IMARIS analysis the robust protein increase was mainly detected on primary afferent axontermini and GFAP-labelled astrocyte membrane compartments within the spinal dorsal horn, but not on postsynaptic dendrites was also validated ultrastructurally. Furthermore, lumbar DRG analysis demonstrated that peptidergic and non-peptidergic nociceptive subsets of ganglia cells were also abundantly positive for P2X4 receptor in CFA model.
Conclusions
Here we provide novel evidence about involvement of neuronal and glial P2X4 receptor in the establishment of inflammatory pain.