Acupuncture Regulation On Hypothalamic Gonadotropin-releasing Hormone Neurons

1 ObjectiveThe experiment is to study the most effective acupoints to treat genital-related diseasesand the regulation of different stages of the estrous cycle on genito-endocrine function byacupuncture stimulation. The study can offer experimental evidence to enhance acupunctureeffect and consummate mechanism, and direct standardization of acupoint selection methodtreating genito-endocrine diseases by acupuncture.2 MethodExperiments were performed in 260 virgin female Sprague-Dawley rats and normalestrous cycle weighing between 240 and 280 g. In the morning of the experiment day, a dropof female rat vaginal secretion repeated dilution with saline was imbibed in the vaginal orificeand HE stain was performed after exsiccation of imbibition in the slide. The estrous cycle wasclassified on the basis of the cells cast-off. The rats were initially anesthetized with anintraperitoneal injection of urethane (1．0-1．2g.kg^-1 ). The experiment coved four parts.Unit recordings: Spontaneous discharges from individual single neurons encounteredwithin the medial preoptic area, the arcuate nucleus and the paraventricular nucleus wererecorded extracellularly with the use of glass microelectrodes filled with a solution of 1．0 MNaCl and pontamine sky blue (tip impedance 10-20 MΩ).①If sexual stimulation induced excitatory or inhibitory responses of the hypothalamicdefined neurons higher than 100%, a feedback intervention test was performed in methods ofintravenous transfusion ofβ-estradial (30-50ng/kg), testosterone (8mg/kg) or hydrocortisone(20mg/kg), individually.Consequently, these neurons were belonged to hypothalamic neurosecretory cells, wewill focus to study the defined gono-neuronal group.To certify the cellular histochemical properties of recording units, in the last studiedneuron of every experimental animal, recording micropipette electrodes with 0．5～1μm glasscapillary (impedance 20-40 MΩ), filled with10% HRP /1 M KC1, after a unit has been isolated and characterized electrophysiologically, HRP is deposited at the recording site withthe following iontophoresis parameters: 50 V (positive) pulses square wave at 10 Hz, 30 min,for further immunohistochemical analysis of labeled neurons.The neurons were marked with HRP for survival times of 3h and followed by TMBstaining and immunohistochemical analysis of GnRH to investigate the relation between therecorded neurons and GnRH neurons.We regard exciting genital-related neurons in hypothalamus as the standard, comparingby acupuncturing 8 acupoints of different parts of the body for 30 seconds, to observe if thereis any differences in exciting activity magnitude of the hypothalamic neurons to identify themost effective parts and acupoints corresponding different phases of the estrous cycle.②This experiment has two groups: the first group is injected by 0．08ml(10%) mustardoil in rat's left ovary; the other group is injected by 0．9% Nacl. In the first group we make amodel of inflammatory ovaries pain. To check the reaction points of plasma exuding withevens blue 0．25ml and compare them to those of the other group.③The adult female SD rat were divided into a control group, Guanyuan(CV4) group andNeiguan(PC6) group according to 4 stages of the estrous cycle. Electroacupuncture(EA)(20/60Hz,2-3mA) was given from 9:30AM to 10:00AM and the brain tissue sampleswere collected at 15:00PM. GnRH expressions in the nuclei related with genitalneuroendocrine in the hypothalamus were investigated with immunohistochemical method.④Adult fem rats were randomly divided into control group Guanyuan(CV4) group andNeiguan(PC6) group according to 4 stages of the estrous cycle. EA was given from 9:30AMto 10:00AM. Blood samples were collected from the common iliac vein before, 0min and 30min after EA for detecting serum gonadotropin-releasing hormone(GnRH) and luteotropichormone(LH) contents with radioimmunoassay.Other female rats were randomly divided into median eminence(ME) lesions group andcontrol group, which were used to observe the influence of hypothalamic ME lesion on theeffect of EA of CV4 in regulating GnRH, LH and E2 levels.3 Results①We have recorded the genital-related neurons inside the medial preoptic area, thearcuate nucleus and the paraventricular nucleus in hypothalamus. There were more recordedneurons in the medial preoptic area than in the arcuate nucleus and the paraventricularnucleus. Depending on the results of marks with HRP and immunohistochemical analysis ofGnRH, the conclusion is that the marks of the neurons and GnRH-positive reactants almost overlap each other.②According to the dorsal ganglion of acupoints of acupuncture from up to down asthe following sequence the increased percentage of the exciting activity excitability of theneurons which is genital-related neurons in hypothalamus: Neiguan(PC6) , Shanzhong(CV17) ,Junynshu(BL14) , Zhongwan(RN12) , Shenshu(BL23) , Guanyuan(CV4) , Zigong(Ex-CA1) ,Zhusanli(ST36) : 122．59%, 124．68%, 205．67%, 204．68%, 246．58, 341．76%, 444．15%,291．57% spikes/s in estrus; 77．85%,107．81%,117．37%,202．39%,212．39%,473．24%,450%,299．68% spikes/s in anestrous.The percentagte in anestrous approaches or exceeds that of estrum. According to thedifferent parts of acupuncturing, the augmented percentage decreases as the followingsequence : low abdominal, low tergal and crural area, epigastric zone and back, thoracic part,upper limb and face.③The points of evens blue-skin correspond comparative intensive area with acupointsarea: Guanyuan(CV4) -Zigong(Ex-CA1) acupoints area, Shenshu(BL23) -Mingmen(DU4) acupoints area, Changqiang(DU1) -Huiyin(RN1) acupoints area, Sanyinjiao(SP6) -Zusanli(ST36) acupoints area, Zhongwan(RN12) acupoints area, Ganshu(BL18) acupoints area.④The result of the immunohistochemical staining shows the number of theGnRH-positive reactant are differently in the medial preoptic area, the arcuate nuclei, thenuclei periventriculares, the septal area and the nucleus amygdalae in different phases ofestrous cycle in the control group, with more expression in the proestrus and estruation. Afteracupuncture, the GnRH expressions in the medial preoptic area, the arcuate nuclei, the nucleiperiventriculares, the septal area and the nucleus amygdalae were increased in different stagesof the estrous cycle in varying degrees, with the "strong" GnRH expression increasedsignificantly in the diestrus and the metaoestrus in the Guanyuan(CV4) group.⑤:Before the treatment, serum GnRH and LH levels were slightly different in differentstages of the estrous cycle, GnRH level was highest in the estruation and LH content highestin the preoestrus. After EA, both serum GnRH and LH levels increased to a certain degree atmost stages. Compared with control group, GnRH levels in the diestrum and metaoestrus andLH in all the 4 stages of the estrous cycle in CV4 group increased significantly (P<0．05,0．01) .Self comparison showed that after EA of CV4, serum GnRH level in diestrum and metaoestr-um, and LH contents in diestrum and metaoestrus increased markedly (P<0．05,0．01) ; whileafter EA of PC6, serum GnRH and LH levels in diestrum and metaoestrum increased slightly,suggesting that the effect of EA of CV4 was superior to that of EA of PC6 in promotingsecretion of GnRH and LH. Thirty minutes after EA, self comparison between pre andpost EA in each group and comparison among 3 groups showed no significant differences in both serum GnRH and LH levels (P>0．05) . Electrolytic lesion weakened the effects of EAin promoting the secretion of GnRH, LH and E2, though the influence of this lesion on GnRHsecretion was milder.4 Conclusion①The electrophysiological method adopted in our experiment is a simple and rapid wayto identify genital-related neurons in hypothalamus in vivo.Depending on the results of marksof HRP and immunohistochemical analysis of GnRH, the neurons were suspected to be theGnRH neurons.②Acupuncture can effectively regulate the activation of the genital-related neurons inhypothalamus. But the regulating intensity is different from various acupoints stimulated. Theeffect Zigong(Ex-CA1) , Guanyuan(CV4) , and Zusanli(ST36) acupoints is stronger for studyof 8 acupoints.Stimulating the acupoints or parts innervating by the same spinal cord segments with thegenital organ can produce the most effection activating the neurons. The increased percentageof the exciting activity excitability of the neurons in anestrous equals or exceeds that ofestrum's rats.③The reaction points of plasma extravasation resulting from the inflammation ovaries isidentical with the ovary's corresponding dorsal ganglion. The acupoints have specificregulating function to the splanchna with the reaction points corresponding by EA stimulated.④After EA, the expression of GnRH-positive reactant in the medial preoptic area, thearcuate nuclei, the nuclei periventriculares, the septal area and the nucleus amygdalae of thehypothalamus significantly increases, with the Guanyuan(CV4) group better than theNeiguan(PC6) group, and more obvious expressions in the diestrus and the metaestrus.⑤EA can enhance the secretion of GnRH and LH in the diestrum and metaoestrus of theestrous cycle, which is realized by the hypothalamus pituitary gonad axis.The effect of EAis to promote the excreting of GnRH and enhance its level, which is mainly realized throughhypothalamus pituitary-gonad axis.