| The pain is an unpleasant and intolerable feeling resulting from existing or potential tissue damage. IASP defined neuropathic pain as the pain caused by lesion or disease of the nervous system. Neuropathic pain features spontaneous pain, hyperalgesia, pain hypersensitivity. The high incidence of the neuropathic pain results in the body and psychology suffering of patients and reduces their life quality seriously.The trigeminal neuralgia is one type of the neuropathic pain. It means the recurrent, momentary, fierce pain which occurs in the region of the trigeminal nerve. The mechanism of the trigeminal neuralgia is still unknown although scientists performed large amount of observation and experiments. The theories which explain the trigeminal neuralgia more comprehensively and accepted widely are the central theory, the peripheral theory, the immunological theory and the neuropeptide theory.There is still lack of satisfactory treatment and medicine for the neuropathic pain clinically. Therefore, it is necessary to develop new medicine and explore the mechanism. CQM is a prescription formulated by our research group. It has functions of promoting blood circulation and Qi, expelling wind and removing dampness, activating meridians to stop pain. In our previous study, CQM evinced superior analgesia to several neuropathic pain models.This experiment is planned to adopt photochemically-induced ischemic IoN injury rat model to observe the analgesic effect of CQM; free and awaking animals were sampled by microdialysis, combining with HPLC-FLD technology, to observe the impact of CQM on the amino acid neurotransmitters and to explore the central pharmacological analgesia mechanism of CQM.Methods1Rats adapting stimuli, grouped and modelingAll the rats were stimulated by Von Frey hairs5times a day, until they reacted calmly before the experiment carried out. Rats whose mechanical allodynia were above26g on three consecutive days were divided into the operation group and the sham group randomly. Photochemically-induced ischemic IoN injury surgery was carried out on the operation group. The method of the surgery in detail was to expose the IoN, then inject erythrosine B i.v. and put the rat under the laser machine. Make sure the laser spot on the nerve exactly. The sham group accepted the surgery of exposing the nerve then sewing the skin up. On three consecutive days after the modeling surgery the mechanical allodynia was above26g indicated the operation was successful. The successful rat models were divided into the model group, gabapentingroup(i.p.100mg·kg-1).CQM-L group(i.p.35mg·kg-).CQM-H group(i.p.70mg·kg-1),model group and sham group i.p. normal saline of same volume.2Measuring methodsRats were in small box for ten minutes to let them get used to the surroundings. Rats were stimulated with Von Frey hairs from0.008g to26g, once a second.3positive reactions in5stimuli indicated the minimum value was the mechanical allodynia. The positive reactions are attacking, withdrawing and scratching.3Drug administration and the effectThe basic time point is Omin. We measured the IoN region30,60,90,120,180,240and300min after the i.p. drug administration.4MicrodialysisAfter the drug administration and measuring, anaesthetized the rat then performed the brain surgery of implanting the probe casing pipe into striatum. The probe was implanted into the pipe when the rat woke up at the first day after the surgery. Perfusion of the tube system with compound sodium chloride2ul·min-1was carried out after the probe implant. Samples were taken every30min after60min of stabilization. The average value of the first two samples was the basic level of Omin. We examined the Glu, GABA and energy metabolism related substances30,60,90,120,180、240、300min after the drug administration.5HPLC-FLD examined Glu, GABA levelChromatographic column was Eclipse AAA4.6mm×150mm,5μm. Derivative solution was120μl methanol mixed with5mg OPA,10μl beta-mercaptoethanol and 0.2mol·L-1boric acid buffer solution (PH is9.2). The mobile phase included solution A and B, the buffer solution was20nmol·L-1sodium acetate, pH7.2. OPA automatic precolumn was derivatizated with340nm excitation wavelength and455nm emission wavelength.6The ISCUS examined the energy metabolism related substancesPut glucose, lactate, pyruvate, glycerol agents into the ISCUS in order. Put samples in after the ISCUS carried out the automatic calibration to examine the level of the energy metabolism related substances.Results1The trigeminal neuralgia rat model induced by photochemical ischemic IoN injury51rats that met the inclusion criteria after adaptive stimulation received photochemical modeling surgery. According to the evaluation results of the mechanical pain threshold of the trigeminal nerve facial dominating areas, it was determined that32rat models were successful, with a rate of63%.The mechanical pain threshold of trigeminal neuralgia rat model decreased significantly after the modeling, from26g to1.60±1.74g (P<0.01).10days after the trigeminal nerve injury, mechanical pain threshold of the surgical group was1.49±1.67g. The mechanical pain threshold decreased significantly (P<0.01) compared to24.17±4.49g of the control group.This indicated that the model was successful and the behavioral changes of the operated rats were caused by trigeminal nerve injury that induced by photochemical ischemic IoN injury.2Analgesic effect of CQM on the trigeminal neuralgia rat modelThe mechanical pain threshold of the trigeminal nerve dominating areas reflected the efficacy. The results showed that the mechanical pain threshold of surgery group were significantly lower than that of the control group (P<0.05) before administration (0min); after administration (30min), both the treatment group and the model group increased. The CQM high-dose group was significantly higher at three time points (P<0.05), up to23±7.3g; the Gabapentin group was significantly higher at two time points (P<0.05),up to20.5±9.2g;CQM high-dose group was significantly higher than CQM low-dose group (P<0.05) at the point of60min. 3The effect of CQM on Glu, GABA and the energy metabolism related substance in stratum of IoN injury model ratsThe concentration-time area under the curve of Glu, GABA reflects the general level changes of Glu and GABA. Glu of the model group was significantly higher than the sham group (P<0.05). That of all the administration group was significantly lower than the model group (P<0.05). GABA of the model group was higher than the sham group. That of the administration group was lower than the model group. The variance ratio indicates that Glu and GABA is lower in the administration group (P<0.05).The significant relationship among the energy metabolism related substance, the trigeminal neuralgia status and the drug administration was not found.ConclusionsThis experiment adopted the photochemically-induced ischemic IoN injury rat model to observe the analgesic effect of CQM before and5hours after the administration; free and awaking animals were sampled by microdialysis at the same point as behavioral measures, combining with HPLC-FLD technology. CQM was able to release the hyperalgesia behavior of trigeminal neuralgia rats. The mechanism may be related to the reduction of Glu in the striatum. |
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