Prostaglandin E₂ Contributes To Modulation Of Inflammatory Pain In Midbrain Periaqueductal Gray

Pain is defined as an unpleasant sensory and emotional experience associated with actual or potential tissue damage. Pain is a major reason why patients seek medical care in clinics. The treatment of pain, particularly chronic pain associated with inflammatory pain, is at present inadequate. Lack of effective analgesics is partly due to the fact that pain signaling mechanisms are still not fully understood. Therefore, it is important to investigate those aspects in development of therapeatics on pain. Tissue injury or inflammation induces the production and release of a number of chemical mediators including bradykinin, serotonin and PGE₂, which can originate locally or from cells that infiltrate the site of inflammation. These chemical mediators contribute to changes in vascular permeability, resulting in erythema and edema. The inflammatory mediators also sensitize peripheral nociceptors by initiating a cascade of events that change ionic conductances of the nociceptor peripheral terminal, and alter central nervous system related to regulation of pain. Recent evidence indicates that PGE₂ is critical for the processing of pain not only by sensitizing peripheral terminals of primary afferent nociceptors but also by modulating of pain processing at central nervous system.Mostly, pain information begins at simple, naked nerve endings called nociceptors that form a functional pain unit with nearby tissue capillaries and mast cells. Tissue injury causes these nerve terminals to depolarize, an event that is propagated along the entire afferent fiber eventuating in sensory impulses reaching the spinal cord. Impulses arrive at the cell body in the dorsal root ganglion and travel along projections to secondary afferent nerves in the dorsal horn. Impulses then cross to spinal thalamic tracts on the opposite side of the cord before ascending to the thalamus where pain begins to become a conscious event.In central nervous system, there is a nerval network system to regulate nociceptive signals. A pathway from the midbrain periaqueductal gray (PAG) through the rostral ventromedial medulla (RVM) to the dorsal horn constitutes a putative endogenous nociceptive modulatory system. The PAG is an important site in ascending pain transmission. It receives afferents from nociceptive neurons in the spinal cord, and sends neuronal projections to thalamic nuclei that process nociception. Activation of this system inhibits nociceptive neurons in the dorsal horn of the spinal cord. The periaqueductal gray (PAG) refers to the region of the midbrain that surrounds the cerebral aqueduct. The PAG is subdivided into four regions, dorsal, dorsolateral, ventrolateral, and ventral subdivisions. The PAG is also a major component of a descending pain inhibitory system.At the site of inflammation, PGE₂ sensitizes peripheral nociceptors through activation of EP receptors present on the peripheral terminals of sensory neurons by reducing pain threshold and increasing responsiveness. PGE₂ is also produced in the spinal cord after tissue injury, where it increases excitability of the spinal cord dorsal horn neurons that produces pain hypersensitivity.Arachidonic acid, the precursor of PGE₂, is stored in phospholipids on the plasma membrane and cleaved by phospholipase A2 on the receptor-mediated or receptor-independent stimulation. The initial step in the biosynthesis of PGE₂ is the metabolic conversion of arachidonic acid to PGH2 via PGG2, which is the common precursor for the synthesis of prostanoids. PGH2 is converted to PGE₂ by microsomal PGE synthase. COXs are rate-limiting enzymes in the arachidonate cascade. COX-1 is a constitutive form that is expressed in almost all tissues, including stomach, kidney, and platelets, and considered to modulate physiological responses. On the other hand, COX-2 is induced in various cell by cytokines, hormones, and mitogens and accounts for PGE₂ production in the course of inflammation. In the basal state, PGE₂ is considered to be formed and inactivated within the same cell or neighboring cells prior to their release into circulation as inactive metabolites. It is well established that the half-life of PGE₂ in circulating blood is very short and it has been reported that only 3.2% of the total injected remains after 90s. Numerous studies indicate that PGE₂ in circulating blood is unable to cross the blood-brain barrier and penetrate the brain. Many studies have reported that the cytokines that have received most attention as important for inflammatory information-to-brain communication are interleukin-1β. Dual-labeling in situ hybridization histochemistry demonstrated that the vast majority of the COX-2-expressing vascular cells also responded with an induction of mPGES in response to IL-1β,and that the mPGES mRNA-expressing cells co-expressed IL-1βreceptor subtype 1 mRNA. This suggested that single cerebrovascular cells possess the components necessary for the production of PGE₂ from arachidonic acid on IL-1βstimulation. PGE₂ produces a broad range of biological actions through their binding to sepcific receptors on plasma membranes. In situ hybridization and immunohistochemical studies have provided detailed information on brain distribution and expression of PGE₂ receptors. Four subtypes of PGE₂ receptor( EP1-4 ) have been classified. EP1 receptors couple with the Gq-phospholipase C-IP3 pathway. EP2 and EP4 receptors couple with the Gs-adenylyl cyclase-cAMP pathway and activation of those receptors increases cAMP levels. In contrast, activation of EP3 receptors mainly inhibits cAMP generation via Gi coupled mechanisms. EP3 has been reported to mediate a number of the physiological functions of PGE₂ in the CNS such as pain modulation and regulation of the autonomic nervous system. EP3 mRNA is widely expressed over the CNS including ascending nociceptive pathways and descending modulatory pathways. EP3 is also expressed by IL-1-responsive neurons in nociceptive pathways. EP3 receptors have specifically been localized in the PAG.Glutamate and GABA are the main neurotransmitter in the CNS, and mediate exciatory and inhibitory activities of neurons. Among regions of the PAG, the dorsolateral (dl) region receives abundant somatic afferent inputs from the dorsal horn of the spinal cord and also sends descending neuronal projections to the medulla in regulating pain and autonomic activity. Glutamate, the major excitatory neurotransmitter, appears in the dl-PAG region. The dl-PAG also has the high density of excitatory amino acid binding sites (glutamate receptor subtypes) including non-NMDA, NMDA and metabotropic receptors. In addition, GABA-mediated neuronal elements constituting ⁵0% of the total population of neurons play a crucial role in the intrinsic neuronal circuitry of the PAG. The GABA synaptic inputs make up ⁵0% of the synaptic innervation of the PAG neurons and the majority of GABAergic neurons are tonic active interneurons. The release of GABA from those neurons may play a role in modulation of the synaptic inputs to the PAG neurons. Studies have further shown that GABAA receptors are dense within the PAG. A prior study has shown that cyclooxygenase inhibitor injected into the PAG attenuates pain response from cutaneous and visceral afferent nerves. Microinjection of PGE₂ into the PAG facilitates nociception through descending activation of the rostral verntromedial medulla. However, a mechanism by which PGE₂ within the PAG induces hyperalgesia is unclear.In 1976, Neher and Sakmann developed a patch clamp recording technique. This recording technique has been used to record ion currents activity on single/mutiple channel on neurons to reflect molecular events of neurons. The principle of this technique is to record currents by using a tight giga-ohm seal between glass recording pipettes and patch neuron. When whole cell access is established, whole cell voltage-clamp recording is performed to obtain excitatory and inhibitory postsynaptic currents (under holding current=-70mv). The excitatory and inhibitory postsynaptic currents represent the synaptic quanta release of glutamate and GABA that play a role in modulating the activity of the postsynaptic neuron. A whole cell current-clamp technique is used to record the spontaneous firing activity (under holding current=0mv). The change of action potential represents the neuronal activity. By using in vitro brain slices preparations and patch clamp recording technique, we can confirm a mechanism by which PGE₂ modulates neuronal activity in the PAG via synaptic transmission.In this study, we did the following experiments:1. CFA injection into the hindpaw of rats was used to induce inflammatory pain model. Inflammation and swelling of the injected paws were apparent after treatment with CFA. Inflamed paw was larger than the contralateral non-inflamed paw. All experiment animals experienced increased nociceptive input (e.g. limping, decreased locomotion activity, guarding). This inflammatory pain model laid the foundation for the further experiments.2. A microdialysis probe was stereotaxically implanted into the PAG. The dialysis system was attached to a pump and PGE₂ samples were collected. 3. PGE₂ concentrations in the dialysate samples were determined using a PGE₂ EIA kit.4. Using immunohistochemistry and fluorescent labeling technique to detect EP3 receptor express. Our results show that inflammatory pain greatly increased the EP3 receptor expression in PAG.5. Whole cell voltage-clamp recording was performed to obtain excitatory and inhibitory postsynaptic currents of the dl-PAG neurons. After bath application of PGE₂, the effects of PGE₂ on frequency of mEPSCs and mIPSCs were recorded. Our results show that PGE₂ inhibits glutamatergic synaptic transmission in dl-PAG via activation of presynaptic EP3 receptors.6. Spontaneous action potential of the dl-PAG neurons was recorded using whole cell current-clamp methods. PGE₂ significantly attenuated the discharge rate of the dl-PAG neurons. The decrease of firing activity was abolished in the presence of glutamate NMDA and non-NMDA receptor antagonists.From these results we can conclude that inflammatory pain greatly increases the PGE₂ production, and thus EP3 receptor expression in PAG. These results support the concept that PGE₂ involves in modulation of inflammatory pain in PAG. PGE₂ inhibits glutamatergic synaptic transmission in dl-PAG through activation presynaptic EP3 receptors. In contrast, PGE₂ had no distinct effect on GABAergic synaptic transmission. This suggests the lack of PGE₂ effects on the synaptic GABAergic terminals in the dl-PAG. In addition, spontaneous action potential of the dl-PAG neurons was recorded using whole cell current-clamp methods. PGE₂ significantly attenuated the discharge rate of the dl-PAG neurons. The decrease of firing activity was abolished in the presence of glutamate NMDA and non-NMDA receptors antagonists. The results from the current study provide the first evidence indicating that PGE₂ inhibits the neuronal activity of the dl-PAG via selective attenuation of glutamatergic synaptic inputs, due to activation of presynaptic EP3 receptors. This new mechanism opens a new avenue for research aimed at the development of new anti-inflammatory pain drugs. We have confidence that the antagonists of EP3 receptors and glutamate NMDA and non-NMDA receptors will become potentially the new targets to treat inflammatory pain.The features and innovations:1. This study revealed a mechanism by which PGE₂ is involved in the modulation of inflammatory pain in PAG.2. A microdialysis probe was stereotaxically implanted into the PAG to collect dialysate samples. PGE₂ concentrations in the dialysate samples were determined. This method is more specific than collecting cerebrospinal fluid.3. Patch-clamp technique was used to determine the role of PGE₂ in modulating neuronal activity of the PAG through excitatory and inhibitory synaptic inputs.4. This new mechanism opens a new avenue for research aimed at the development of new anti-inflammatory pain drugs by developing antagonists of EP3 receptors and glutamate NMDA and non-NMDA receptors.