| Numerous research showed that the perception of pain is highly complex, and requires neural integration from a variety of routes, these pain dimensions and their interactions relate to cortical and other subcortical brain structures that processes nociceptive information both in parallel and in series, which were processed, coded and regulated together. The new nociceptive research mode was transferred from single target to neural network associated with the sensory, affective and cognitive dimensions of pain (NNSACP). Headcache, as the central neuropathic pain, is related to percipience, cognition and mode, on the other hand, it is well known that synaptic plasticity is related to pain. Therefore, hippocampus, as a major component of limbic system, which plays a key role at learning and memory and mode, it is also important at pain related signal transduction pathway and involved in nociceptive signal processing, hippocampal pyramidal neurons respond to varies harmful stimuli. Researchers found that pain induced morphological and physiological alteration at hippocampus and affected its learning and memory and mode function.Therefore, we focused on the effect of Zhengtian pills (ZTP) on hippocampal synaptic pathway, included perforant path-to-dentate gyrus-to-CA3-to-CA1, which was called the trisynaptic circuit. The goal of the present study is to investigate the molecular mechanisam of ZTP on headache from the different direction. The present study consists of four major parts:Part 1 Effect of ZTP on the behavior of headache induced by nitroglycerin in ratsObjective To investigated the effect of ZTP on the general behavior and symptoms of nitroglycerin-induced in rat model of headache.Methods All rats were randomly divided into control, positive control, migraine model, low dose and high dose (0.54,1.08 g/kg) of ZTP administration groups, nitric oxide donor-nitroglycerin was injected to induce migraine headache. The time of ears turning red, frequency of scratching head, climbing the cage and head-twitching were used to evaluate rat behaviors and effect of ZTP on headache parameters in rats was determined.Results 2 minutes after nitroglycerin injection, rats showed headache phenomena that parallel clinical symptoms of migraine, which peaked in 30 min, and lasted for 60 min. Frequency of head-twitching and the numbers of scratching head with fore-limb and hind-limb GTN group rats were significantly increased. In contrast, after ZTP (1.08 g/kg, p.o.) treatment, the numbers of scratching head with fore-limb, hind-limb and the frequency of head-twitching were significantly decreased. Flunarizine, as the positive control of ZTP and low dose group ZTP (0.54 g/kg) also showed a trend to decrease the numbers of scratching head and head-twitching frequency with fore-limb and head-twitching, but no meaning of statistical significance. The numbers of climbing the cage of ZTP (0.54,1.08 g/kg) and Flunarizine groups were not significantly decreased.Conclusion The present study showed that the headache model had established successfully by subcutaneous nitroglycerin injection and ZTP could significantly improve symptoms of headache in rats.Part 2 Effect of ZTP on synaptic transmission in perforant path-dentate gyrus (PP-DG) pathway of anesthetized ratsObjective To investigate the effect of ZTP on synaptic transmission in PP-DG pathway of anesthetized rats.Methods The extracellular recording techniques in vivo were used to investigate the effect of ZTP on synaptic transmission in PP-DG pathway. Rats were anesthetized with urethane carbamate before being fixed in a stereotaxic frame. The holes were sequentially drilled for a monopolar recording electrode and a bipolar stimulating electrode respectively. The stimulating electrode was lowered into the perforant path and the recording electrode was placed in the granular cell layer of dentate gyrus. An evoked response was generated by test stimulation (0.033 Hz) with single constant current pulses. After ensuring a steady state baseline response, which was taken about 30 min, LTP was induced using a high-frequency stimuli protocol to investigate the effect of ZTP on synaptic transmission in PP-DG pathway of anesthetized rats.Results Compared with the control group, the population spike amplitude (P< 0.05) was significantly reduced in GTN group, which of the ZTP (0.54,1.08 g/kg) and Flunarizine groups were significantly improved compared with GTN group (P< 0.001, P< 0.01, P< 0.05).Conclusion Our results showed that ZTP improved synaptic transmission in PP-DG pathway of anesthetized rats, which may play an important role in the pathogenesis of headache.Part 3 Effect of ZTP on synaptic transmission in Schaffer Collateral-CA1 pathway of rat hippocampal slicesObjective To investigate the effect of ZTP on synaptic transmission in Schaffer Collateral-CA1 region of rat hippocampal slices.Methods The brain block was cut with a vibratome into 350μm-thick transverse slices in ice-cold artificial cerebrospinal fluid (ACSF). Using the extracellular recording techniques in vitro, a stainless steel concentric bipolar electrode was inserted into the Schaffer collaterals fibers and the recording electrode was inserted into CA1 region close to the granular layer. Hippocampal slices were bathed in ACSF bubbled with 95% O2 and 5% CO2. Signals were amplified with Axon Digidata 1440A amplifier, recorded using AxoScope acquisition software and analyzed with the Clampfit 10.2 software package to investigate the effect of ZTP on synaptic transmission in Schaffer Collateral-CA1 region of rat hippocampal slices.Results Compared with control group, fEPSP slope was significantly reduced in GTN group (P< 0.05), which in the ZTP (0.54,1.08 g/kg) and Flunarizine groups were obviously improved compared to the GTN group (P< 0.001, P< 0.01, P< 0.01).Conclusion Our results showed that ZTP significantly improved synaptic transmission in Schaffer Collateral-CA1 region of rat hippocampal slices, which may play an important role in the pathogenesis of headache.Part 4 Effects of ZTP on TRPV1 expression in rat hippocampus and cortexObjective To investigate the effects of ZTP on TRPV1 expression level and explore the molecular mechanism of its efficacy in headache.Methods All rats were randomly divided into control, migraine model and high dose (1.08 g/kg) of ZTP administration groups, nitric oxide donor-nitroglycerin was injected to induce migraine headache. After 10 days administration of ZTP, and then dissecting the cortex and hippocampus from the brains, the transient receptor potential vanilloid (TRPV1) receptor expression level both in cortex and hippocampus was determined by western blotting.Results ZTP significantly decreased the up-regulated TRPV1 protein expression level both in cortex and hippocampus induced by nitroglycerin.Conclusion TRPV1 might be one of the important molecular mechanisms of its efficacy in headache. This is the first report about the effect of ZTP on TRPV1 protein expression level. |
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