Expression Of CB1Receptor In Rat Model Of Trigeminal Neuropathic Pain

1. BackgroundCannabinoid receptors described to date belong to the seven-transmembrane-domain receptor superfamily and consist of two major cannabinoid receptor subtypes, namely cannabinoid-1(CB1) and cannabinoid-2(CB2) receptors. CB1receptor is constituted of473amino acids and7transmenbrane domains, and CB2receptor of360amino acids and transmembrane domains. They are G protein-coupled receptors. Cannabinoid (CB1) receptors are found predominantly in the brain with highest densities in hippocampus, cerebellum and striatum and mainly distributed in presynaptic membrane of never terminals and regulating the release of neurotransmitters. Locations outside the central nervous system have also been described for CB1, for example, in the uterus, testis, adrenal gland, heart, lung and ovary. The second cannabinoid subtype CB2receptor is predominantly expressed in the immune sysem such as spleen, tonsil, monuclear cells, T cells, and B cells and macrophages and so on, palying important roles in immune sisterm regulation and inhibiting the release of cytokines. Reports say the CB1agonists can produce good analgesic effect and inhibit the excitability of pain neurons. The CB1antagonists enhance the acity of pain neurons and enhance the sensitivity of the hamful stimulus. CB1receptors can restrain hamful pain and reduce pain related spontaneous behavior.Trigeminal neuralgia is beginning paroxysmal and clicks to like some ache in the trigeminal nerve distribution area, it lasts seconds to minutes. There are asymptomatic in interval.Pain may be due to any oral or facial stimulation. It commons in elderly and mostly is unilateral. So far, the pathogenesis of TN remains unclear, the exact model is difficult to establish. Domestic and foreign experts believe that surround factor is the main pathogenic factor of TN. Most experts agree that the vascular compression theory in the surround factors is the main pathogenic factor. Therefore, most animal models simulated by ligation of the infraorbital nerve vascular compression, and stimulate the surface of the skin by dominating the infraorbital nerve to simulate of the trigger points of trigeminal neuralgia.Spinal trigeminal nucleus caudalis subnucleus (Vc) is a portal that nociceptive information transmits from peripheral to the central. There is a variety of terminal of neuropeptide-positive fibers which conduct pain in Vc. Most of them derived from the trigeminal ganglion neurons, and formed of synaptic connections with a large number of cell bodies and dendrites that distributed in the superficial layers of Vc neurons. So we test expression and activity of the CB1in Vc tissue.Some research results showed that the expression of μ-opioid receptor in rat and monkey dorsal root ganglia neurons decreased. On the contrary, after unilateral nerve injury, the expression of CB1receptors on thalamus, and its expression was also raised in the sciatic nerve choronic constricition injury(CCI) in the superficial spinal cord of rats with dorsal angle. After harmful and harmless stimulus, rats’snout in the unilateral inferior alveolar nerve transaction in rats can induce Fos and cdk5/p35protein-like immune responses in bilateral trigeminal spinal tract nucleus(victims of spinal injuries trigeminal nucleus of Vc). Vc, as a sensory relay station nuclear, was accepted head faical sensory information coming from trigeminal nerve. The cell and molecular biology mechanism of CB1receptor’ expression increasing is not clear now. In the sciatic nerve CCI moderl, the tyrosine kinase receptors and intracellular mitogen-activated protein kinase and protein kinase C may be involved in the regulation of CB1receptors after CCI.In conclusion, we tested the expression of CB1in a rat model of trigeminal neuropathic pain and effect of CB1receptor in ION-CCI rat model.2. ObjectiveWe according to CB1receptors primary trigeminal neuralgia in the rat model of neurons in Vcand the sensory nerves of trigeminal nerve afferent the lower medulle oblongata of the Vc neurous. So we consider making the ION-CCI rat as experimental study model of the trigemninal neuropathic pain, and test the expression of CB1in the Vc and the effect of CB1receptor in ION-CCI rat model.3. Materials and methods3.1Animal groupsFifty-four male Spague-Dawley rats that weight300-350gram with loosely ligated unilateral nerve were randomly divided into six groups:normal group, sham group, ION-CCI-1d, ION-CCI-3d, ION-CCI-7d, ION-CCI-14d. They were housed in plastic cages (46cm×24cm×20cm; three or four rats per cage) under a partially reversed12:12hr dark/light cycle (lights on at1500hr) in a colony room with an ambient temperature of22-25℃and humidity40%-50%. Water and food were available ad libitum in the animal laboratory of zhujiang hospital.3.2The ligation of infraorbital nerveThe normal group without any treatment, the sham group just exposed the infraorbital nerve. Rats were anesthetized with chloralic hydras (350mg/kg, i.p.). All surgery was performed under direct visual control using a operation microscope or naked-eye observation. The sham group just exposes the infraorbital nerve, and then suture the wound, the other operation groups exposed one side of infraorbital nerve and ligate it. But it must maintain the neural microcirculation, and then suture the wound.3.3Preparation of infraorbital nerve and Vc tissue.Weigh the rats according to the groups; narcotize the rats by intraperitoneal injection of chloralic hydras (350mg/kg, i.p.). Taking the nerve tissue of infraorbital nerve ligation area to make the HE and WEIL stains, then observed. Cutting the rats’Vc tissue according to Paxinos and Watson rat brain stereotaxic map. Put the tissues into the test tubes then store in the-80℃fridge3.4The extraction of antigen protein of Vc tissueTaking out the tissue from the marked test tubes, then put them into homogenizer, after added the cell lysis solution and placed the homogenizer on ice and thoroughly homogenized, make the liquid of after homogenated into centrifuge tubes. At4℃and12000rpm, pick up the supernatant to the marked test tubes and put inside the-20℃fridge after centrifuged5minutes. 3.5Test the protein content of supernatant of Vc tissue.3.6Test the western blotting of CB1according to the measured protein content.3.7Analyse the effect of CB1receptor in ION-CCI rat model.3.8All the rats’ responses to VON FREY stimulate threshold and globe threshold were measured.4、Statistical method and statistical softwareExperimental data were analyzed by using SPSS13.0. with average standard deciation said. One-way ANOVA and Repeated Measures were performed on data. First we test Homogeneity of variances. If P>0.05, using Bonferroni test to compare between groups. If p<0.05, using Dunnett’s test to compare between groups. Comparison between two groups with Paired-Samples T Test. p<0.05said differences with a statistical significance.5. Results(1) Results of behavior test:No significant difference of pain threshold of postoperatice had found between normal group and sham group(P>0.05). The pain threshold of postoperatice had significant difference in the operation groups(P<0.05).(2) Result of histology observation:The never fiber of narrow ring region after operation swelled gradually. We can see that degeneration of nerve fibers, thickening myelin, the release of lamellar myelin vacuolation, bare axons, the enlarged schawan cells. Longitudinal slices can see the myelin was worm-eaten-like focal rupture missing axion sparse in the eccentric position and the normal structure disappeared.(3) The expression of CB1receptor in Vc tissue was increased successively in ION-CCI Id group, ION-CCI3d group, ION-CCI7d group and ION-CCI14d group with no significant difference. The expression of CB1receptor had no significant difference bwtween the two sides of sham group, while significant difference had found in ION-CCI14d group.6. Conclusion(1)Peripheral nerve damage can be presented in the form of time-dependent leading to upregulation of CB1receptores in the Vc tissue.(2) ION-CCI induced ipsilateral CB1receptors increased and the expression of CB1receptor in the CCI side increased siginicant greater than the contralateral. (3)No direct correlation had found between the expression of CB1receptor and pain threshold of peripheral nerve.