Expression of P2X4 receptor by lesional activated microglia during formalin-induced inflammatory pain

https://doi.org/10.1016/j.jneuroim.2005.03.007Get rights and content

Abstract

P2X4 receptor (P2X4R) is an ion channel gated by adenosine 5'-triphosphate. Here we report the presence and the distribution of P2X4R in rat spinal cord by immunohistochemical analysis in an inflammatory pain model. Peripheral inflammation was induced by subcutaneous injection of 4% formalin into the rat hindpaw. Morphology, spatial localization, and activation state of P2X4R+ cells were described at 1, 5, 7, 14, and 28 days after injury. In normal and saline treated control rats, P2X4R was rarely seen. After formalin administration, an increase of P2X4R+ microglia were observed in the spinal cord dorsal horn on the side ipsilateral to the injection, reaching maximal levels by day 7, and then decreasing to normal levels by day 14. This implicates a role of P2X4R in the spinal inflammatory pain process. Furthermore, formalin-induced region-specific increase in activated microglia was confirmed by ED1 and endothelial monocytes activating polypeptide II (EMAP-II) expression. In conclusion, this is the first demonstration that P2X4R is expressed by microglia in the 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).

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