Expression of P2X4 receptor by lesional activated microglia during formalin-induced inflammatory pain
Introduction
The formalin test is a well-established and frequently used model to study mechanisms of pain. It produces a long-lasting noxious input towards the spinal cord and the brain (Tjolsen et al., 1992). A subcutaneous (s.c.) injection of formalin into the rat hindpaw produces a biphasic excitatory-evoked behavioral response, results in persistent tissue damage, and induces a state with good approximation to some clinical conditions of chronic inflammatory pain (Dubuisson and Dennis, 1977, Tjolsen et al., 1992). Therefore it is widely used to evaluate inflammatory processes, central sensitization, and different pain-mediating substances. Recently, it has been suggested that microglia plays a central role in the development of chronic pain syndromes (McCleskey, 2003). Unlike neurons and astrocytes, microglia are derived from bone-marrow and migrate to the central nervous system (CNS) during development. Under physiological conditions they are resting ramified cells, with many large processes to monitor their environment. When damage to CNS occurs, microglia become activated, adapt a round macrophage-like morphology, and phagocytose both cells and cell debris (Kreutzberg, 1996, Aloisi, 2001). As the strategic cellular sensors responding to a variety of stressors the CNS, microglia orchestrate neuroinflammation by producing cytokines and presenting antigens (Kreutzberg, 1996, Hanisch, 2002). Because they secrete neurotoxic substances, activated microglia has been linked to the pathophysiology of many types of CNS pathologies, including pain (Benveniste, 1997, Hanisch, 2002). In addition to their detrimental activity, microglia can also assume a neurosupportive role by producing neurotrophins (Streit, 2002, Streit et al., 1999). Whether microglia mediate detrimental or beneficial effects probably depends on a variety of factors. Indeed it has been shown that adenosine 5'-triphosphate (ATP) and its purine receptors are involved in the regulation of microglia activation (Ferrari et al., 1997, Hide et al., 2000, Inoue, 2002).
Microglial cells are known to bear two types of purine receptors: ligand-gated (P2X) and metabotropic (P2Y) nucleotide receptors of ATP (Illes et al., 1996). P2Y receptors are responsible for Ca2+ release from intracellular stores. The major physiological mechanism by which activated P2X receptors control cellular functions is the depolarization of the membrane potential and elevation of intracellular Ca2 + concentration in response to extracellular release of ATP, which occurs in neural repair, remodeling and survival, after CNS injury, or acute and chronic diseases (Buell et al., 1996, Volonte et al., 2003). ATP and P2X receptors are most likely to be involved in chronic pain conditions, particularly chronic inflammatory and neuropathic pain (Kennedy et al., 2003). P2X4 receptor (P2X4R) has been reported to be involved in pain signaling in the CNS (Tsuda et al., 2003). As one subunit of P2X receptors, P2X4R mainly interacts with extracellular ATP (North and Barnard, 1997), and is responsible for excitatory neurotransmission to drive many physiological functions, including immune responses and pain (Burnstock, 1999, Di Virgilio et al., 2001). Recently, P2X4R expression by activated microglial cells has been identified as a crucial mechanism in development of neuropathic pain produced by spinal cord injury (SCI) (Tsuda et al., 2003, McCleskey, 2003, Inoue et al., 2004). In general, however, P2X4R expression in the CNS has not been definitively assigned to pathological conditions, particularly in response to peripheral noxious lesion. The sensory neurons for pain, touch, and temperature are distributed in the dorsal horn of the spinal cord. Therefore, it is possible to confirm the existence and size of incoming pain signals by observing the expression of pain related molecules in the dorsal horn after nociceptive stimulation. We have now performed an analysis of P2X4R expression in spinal cord dorsal horn in formalin-induced inflammatory pain.
Section snippets
Animals and treatments
All experiments were done in accordance with the published International Health Guidelines under a protocol approved by the University of Tuebingen Institutional Animal Care and Use Committee and the Administration District Official Committee. Experiments were performed on 8 week old male Lewis rats (195–205 g, Charles River, Sulzfeld, Germany), which were housed at a constant temperature of 22 °C on a 12/12 h light/dark cycle with food and water available ad libitum. Thirty rats were randomly
Results
In the present study, the immunohistochemical distribution of P2X4R was studied in spinal cord tissues at L3–L5 levels of rats with formalin-induced peripheral pain syndrome and in control animals.
Discussion
In the present study, immunohistochemical distribution of P2X4R has been evaluated in the dorsal horn areas in the caudal spinal cord of rat with inflammatory pain induced by formalin (100 μl 4% PFA). P2X4R-IR following right hind paw injection was observed on activated microglia at the ipsilateral side. The kinetics of P2X4R-IR paralleled those of spinal microglial activation in the development of the formalin-induced long-term hyperalgesia (Fu et al., 2001, Yeo et al., 2001), and this was
Acknowledgement
This work has been supported by the DFG (Deutsche Forschungsgemeinschaft).
References (40)
Current status of purinergic signalling in the nervous system
Prog. Brain Res.
(1999)- et al.
Nucleotide receptors: an emerging family of regulatory molecules in blood cells
Blood
(2001) - et al.
The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats
Pain
(1977) - et al.
ATP-mediated cytotoxicity in microglial cells
Neuropharmacology
(1997) - et al.
Microglial reactions after subcutaneous formalin injection into the rat hind paw
Brain Res.
(1999) - et al.
Long-lasting inflammation and long-term hyperalgesia after subcutaneous formalin injection into the rat hindpaw
J. Pain
(2001) - et al.
Expression of P2X(4) receptor in rat C6 glioma by tumor-associated macrophages and activated microglia
J. Neuroimmunol.
(2004) - et al.
ATP- and adenosine-mediated signaling in the central nervous system: chronic pain and microglia: involvement of the ATP receptor P2X4
J. Pharmacol. Sci.
(2004) - et al.
Characterization of a novel tumor-derived cytokine. Endothelial–monocyte activating polypeptide II
J. Biol. Chem.
(1994) Microglia: a sensor for pathological events in the CNS
Trends Neurosci.
(1996)
Depletion of macrophages reduces axonal degeneration and hyperalgesia following nerve injury
Pain
Nucleotide receptors
Curr. Opin. Neurobiol.
Characterization of nociceptive responses and spinal releases of nitric oxide metabolites and glutamate evoked by different concentrations of formalin in rats
Pain
Trophic effects of purines in neurons and glial cells
Prog. Neurobiol.
AIF-1 expression defines a proliferating and alert microglial/macrophage phenotype following spinal cord injury in rats
J. Neuroimmunol.
Reactive microgliosis
Prog. Neurobiol.
The formalin test: an evaluation of the method
Pain
Immune function of microglia
Glia
Role of macrophages/microglia in multiple sclerosis and experimental allergic encephalomyelitis
J. Mol. Med.
P2X receptors: an emerging channel family
Eur. J. Neurosci.
Cited by (67)
Spinal GABAergic disinhibition allows microglial activation mediating the development of nociplastic pain in male mice
2023, Brain, Behavior, and ImmunityThe role of P2X4 receptors in chronic pain: A potential pharmacological target
2020, Biomedicine and PharmacotherapyCharacterization of P2X4 receptor agonists and antagonists by calcium influx and radioligand binding studies
2017, Biochemical PharmacologyPeripheral scaffolding and signaling pathways in inflammatory pain
2015, Progress in Molecular Biology and Translational ScienceCitation Excerpt :For instance, bradykinin differentially activates one of two isoforms of the bradykinin receptor, based upon expression profiles.4 Similarly, several isoforms for the purinergic receptor activated by ATP are also differentially expressed on primary afferent terminals, including P2X2, P2X3,5 P2X4,6 and P2X7.7 However, each of the isoforms in these receptor classes signals through similar downstream cascades.