The Mechanisms Of Central Hypersensitivity Evoked By Glyceryl Trinitrate Infusion In Rats:the Involvement Of Glu-NMDAR-nNOS Pathways

IntroductionMigraine is an incapacitating neurovascular disorder. Most migraine patients exhibit spontaneous pain, hyperalgesia, and cutaneous allodynia during a fully developed migraine attack. There are multiple mechanisms in the pathogenesis of migraine headache, involving arterial vasodilatation, neurogenic inflammation and cortical spreading depression ( CSD). In laboratory animals the disease models are established by either electrical stimulation of trigeminal ganglion (TG) , chemical stimulation of meninges or by the application of chemicals(e. g. K+ ) to the cortex. In this experiment we established migraine models by glyceryl trinitrate(GTN) infusion in rats. GTN infusion induces development of meningeal inflammation, activation of trigeminovascular system, and regional changes in cerebral bood flow( CBF).Head pain in migraine arises within the trigeminal system. Sensitization of central trigeminal neurons accounts for head pain and other symptoms during migraine attack. The trigeminal nucleus caudalis and its caudal extension to the superficial laminae of the C1/2 dorsal horns make up a functional continuum, the trigeminocervical complex (TCC). Neurons in TCC are the major relay neurons for nociceptive afferent input from the meninges and cervical structures; therefore, they are the neural substrates of head pain. Dural stimulation leads to a sensitization of neurons in TCC.Glutamate, a major source of excitatory transmission within the brain and spinal cord, is implicated in cortical spreading depression (CSD) , trigeminovas-cular activation, and central sensitization. N - methyl - D - aspartate receptors (NMDARs) , play an important role in excitatory synaptic transmission, plasticity , and neurodegenation. Furthermore, they implicate in the induction and maintenance of hypersensitivity. Functional NMDARs requires a combination of NR1, an essential channel - forming subunit, and at least one of the NR2 sub-units. The NR2B subunit containing receptors appear particularly important for noception. However, The involvement of glu - NMDAR - nNOS pathways in the development of central hypersensitivity evoked by Glyceryl Trinitrate infusion in rats is still unclear.Materials and MethodsRat migraine model was established with GTN 2μg. kg-1 . min-1 infusion for 30min. The immunohistochemistry staining was used to observe glutamate expression in trigeminocervical complex, and in trigeminal ganglion of rat following GTN infusion or normal saline infusion, respectively. The levels of NR2B, and phospho - NR2B protein expression in cell membrane of rat trigeminocervical complex were detected following GTN infusion for 0.5, 2.0, 4. 0, 6.0, 10. Oh by Western - blot. Western - blot was also used to facilitate the observation of nNOS protein expression in rat trigeminocervical complex following GTN infusion or normal saline infusion.Results1. The glutamate - like immunoreactivity was detected in rat trigeminocer-vical complex and trigeminal ganglion after GTN infusion. The glutamate increased in rat trigeminocervical complex while decreased in trigeminal ganglion.2. The level of NR2B protein expression remain the same after GTN infusion. The increase of phospho - NR2B protein expression in rat trigeminocervical complex was detected after GTN infusion3. nNOS protein expression increase in rat trigeminocervical complex after GTN infusion.