Study On Mechanism Of Peripheral And Central Transmission Of Visceral Function And The Cure Of Electroacupuncture

The disorder of motility of gastrointestinal tract is common in clinical digestive diseases. Its pathogeny and clinical pattern are varied, which are divided into functional and organic. The interdigestive migrating motor complex is the study point as an important motility formation of gastrointestinal tract. In the meantime, the regulation of the acupuncture on the motility of gastrointestinal tract is also interesting. More and more clinical and experimental investigation have shown that electroacupuncture at T Zusanli can relieve the inflammation of gastrointestinal tract; regulate the function of gastrointestinal tract. However, the role of electroacupuncture on MMC and its neurochemical mechanism are still not clear.Mucosal lining and smooth muscle tissue of the stomach are innervated by the autonomic nervous system (ANS) through enteric nervous system (ENS) indirectly. Activation of the parasympathetic innervation increases motility and secretion. The parasympathetic innervation mainly originates in the middle and caudal parts of the dorsal motor nucleus of the nervus vagus (DMV). The neurons in the DMV contain several transmitter types. Besides acetylcholine (Ach), the classical transmitter of the parasympathetic nervous system, the presence of pituitary adenylyl cyclase-activating polypeptide (PACAP), dopamine, and NO in the DMV have been described as well. Many different afferent projection systems have been described to be present in the DMV and influence DMV neuronal activity. Among them are metabolically important peptides like, GABA, TRH, orexin and Substance P. Although the exact regulatory mechanism of the autonomic control of the gastro-intestinal tract is still subject of ongoing investigation, it is well established that the feeding condition is an important factor. Despite the extensive knowledge of the DMV it is at present not known which type of DMV neurons is involved in the gastric response to metabolic challenge.The hypothalamus is the superior central of controlling the autonomous nervous system. It does not only regulate the physiology of stomach, but also regulate other visceral organs as adrenal, liver, heart etc. The regulation of rhythms is involved in importantly. The suprachiasmatic nucleus (SCN), the mammalian central circadian pacemaker, located in the hypothalamus, is responsible for circadian rhythms in physiology and behavior. SCN neurons endogenously display spontaneous circadian rhythms of metabolic and electrical activity in intact animals as well as in vitro, these rhythms have a period of～24h and are reset by light daily. Anatomical studies using transneuronal tracing, revealed polysynaptic pathways between SCN and many organs, such as liver, pancreas, adrenal, pineal and others. Herein the paraventricular nucleus (PVN) was recognized as a critical relay, which contains both preautonomic-sympathetic and preautonomic-parasympathetic neurons.The molecular mechanism of the SCN's clock function is proposed to be composed of several rhythmically expressed genes, consisting of interacting positive and negative transcription/translation feedback loops. However, besides in the SCN, local sets of the clock genes are also rhythmically expressed in peripheral tissues. SCN-lesion and culture experiments on peripheral clock genes have implied that the SCN seems to synchronize these peripheral oscillators. This raises the possibility that SCN efferent may modulate peripheral clock gene expression directly, and subsequently lead to changs in rhythms in both physiology and behavior. Taking all data together, we hypothesize that with change of SCN activity, the synchronized output of the SCN can regulate peripheral circadian oscillations including clock genes, which may be involved in the tissue-specific function.Objectives:1. To investigate the regulatory effects and possible neurochemical mechanism of electroacupuncture on MMC and the changed of AChE and NOS of ENS.2. To determine the expression of several genes, which are known to be present in the DMV projecting to the stomach in response to fasting and refeeding?3. To examine the effect by PVN administration of BIC in the daytime and light exposure in the nighttime on the expression of clock genes (Per1, Per2, Per3, Cry1, Cry2 and DBP) and some tissue-specific physiological related genes in peripheral organs.Methods:1. By provoking rats with pinching their tails continuously the animal model was made. The gastroduodenal motor was recorded by stress gauge. The expression of acetylcholinesterase(AChE) and nitric oxide synthase(NOS) in gastroduodenal myenteric plexus were demonstrated histochemistrically and measured by image analysis system.2. (1) Aglient 2100 bioanalyzer and RT-PCR were used to check the concentration and fragmentation of total RNA from unfixed, temporal fixed and fixed 12h hypothalamus sections; (2)Different neurons of PVN and SON were collected by LMPC, CRH,TRH,AVP,OT mRNA level were measured by RT-PCR; (3)labeled neurons by injecting CTB into stomach and non-labeled neurons in DMV collected by LMPC were checked for house keeping genes by RT-PCR.3. CTB retrograde tracing was used to get labeled neurons in DMV, by isolating labeled neurons and RT-PCR, the levels of transmitters and receptors were measured.4. Under the stimulations of light exposure (day-signal from SCN) and BIC infusion into the PVN (night-signal from SCN), respectively, the mRNA level of clock genes in pineal, adrenal, liver, heart and muscle were measured by RT-PCR.Results:1. (1) The changes of MMC: compared with the control group the cycle, phaseⅠ, phaseⅡelongated (P<0.01), phaseⅢshortened (P<0.01), the frequency and sewing reduced (P<0.01, P<0.05), the incidence rate of phaseⅢdecreased (P<0.01) and the average coordinated contract rate decreased (P<0.01) in the model group. In the gastrointestinal motility disorder groups plus western medicine and electroacupuncture respectively, compared with the model group, phaseⅠ, phaseⅡshortened (P<0.01), phaseⅢelongated (P<0.01), the frequency and sewing elevated (P<0.01, P<0.05), the incidence rate of phaseⅢelevated (P<0.01), and the average coordinated contract rate increased (P<0.01). (2) The expression of AChE: compared with the control group, the model group decreased (P<0.01); compared with the model group, the gastrointestinal motility disorder groups plus western medicine and electroacupuncture respectively increased (P<0.01); (3)The expression of NOS: compared with the control group, the model group increased (P<0.01); compared with the model group, the gastrointestinal motility disorder groups plus western medicine and electroacupuncture respectively decreased (P<0.01).2. (1) unfixed section had higher concentration and better quality of total RNA compared with fixed sections applied in LMPC; relative short amplicons such as GAPDH, NSE, MCH and MC4R were successfully obtained from fixed and unfixed and long amplicon of GR can only be obtained from unfixed material; (2) In magnocellular PVN and SON the expressions of AVP and OT were more special than those in the parvocellular PVN. Oppositely, the expressions of CRH, TRH in the parvocellular were more special than the other two; (3) the expressions of house keeping genes had no significant difference between labeled and non-labeled DMV neurons.3. By fasting, for transmitter the mRNA expressions of PACAP, NOS decreased (P<0.05) significantly compared with control. For receptor, the gene expressions of GABA-AR decreased compared with control. By refeeding, PACAP and GABA-AR recovered as the control level (P>0.05), while the level of NOS still decreased (P <0.05).4. 2hr of bicuculin infusion changed the peripheral clock genes differentially. The expression of clock genes altered in the pineal under both 30 and 60min of light exposure, however only with 60min of light exposure clock genes changed differentially in adrenal, liver and heart. No significant changes can be observed in muscle by 60min of light exposure.Conclusions:1. (1) The dyscoorordination of gastroduoenal motility may play an important role in the pathogenesis of gastrointestinal motility disorder. (2) The decrease of the expression of AChE and the increase of the expression of NOS suggested that the disorders of cholinergic and nitronergic neurons might partake into the neurochemical mechanism of the dyscoorordination of gastroduoenal motility. (3) the act of electroacupuncture on the improvement of coordination of gastroduoenal motility maybe through the cholinergic and nitronergic neurons. 2. The quality and quantity of total RNA from unfixed brain tissues were better than fixed tissues applied in LMPC; LMPC is a novel technique making isolation of homogeneous cells more rapidly in high quality and purity. This could be very useful in the brain because it is divided into numerous small nuclei or irregularly shaped even single neurons to be accurately dissected by hand.3. PACAP, NO and GABA-AR might partake the machanism of regulation of DMV neurons projecting to stomach during fasting and refeeding.4. SCN activity can regulate peripheral clock genes; however, the changes of clock genes were not coupled with the peripheral functions, suggesting the peripheral physiology can happen without the direct involvement of peripheral clocks.