| BackgroundRostral ventrolateral medulla(RVLM) is crucial to the regulation in autonomic and cardiovascular system.The presympathetic neurons in the area mono synaptically innervate sympathetic preganglionic neurons,though which take the control of sympathetic outflow.According to histology,over 40 percent presympathetic neurons are classified into C1 family,which concludes cells containing entire enzyme system for the synthesis of adrenaline.C1 cells have been proven contributing greatly,if not entirely,to the autonomic regulation on both heart and vessels.It must be clear that neither all C1 cells are accommodated in RVLM domain and regulate sympathetic outflow,nor all the presympathetic neurons are C1 cells.So it is not right to assume that presympathetic neurons and C1 cells are the same.Some evident indicated hypertension associated with elevated activity of presympathetic neurons,led by ischemia or hypoxia in brain.Recent researches indicate that hydrogen sulfide(H2S) is a novel gaseous transmitter for its wide spectrum effects under physiological and pathematological status. H2S,highly soluble in aqueous and lipid environments,can readily pass between cells. In physiological solution,the equilibriums H2Sâ†â†’H++HS- and H2Sâ†â†’2H++S2- are rapidly established,and the concentration of H2S and HS- represent 30%and 70%, respectively,of the total sulfide,whereas S2- concentration is negligible.H2S has been detected in micromolar amounts in blood,brain and many tissues. It mainly comes from the metabolic production of mercaptoamino-acid(L-cysteine and homo-cysteine) under cystathionine-γ-1yase(CSE) in peripheral tissues and cystathionine-β-synthet-ase (CBS) in central nerve system.Since H2S is rapidly oxidized,the level of H2S intracellular is controlled by the redox status,oxygen level,as well as dynamic processes of its production.H2S production will increase as homocysteine metabolizes,and the level of the latter is associated with oxidative stress. In peripheral vessel smooth muscle cells,hydrogen sulfide helps nitrogen oxide in the tone regulation though ATP sensitive potassium channel,KATP.The activated KATP hyperpolarizes membrane,closes voltage-dependent calcium channel and leads dilation of vessels.But no reports are found on how hydrogen sulfide on cardiovascular center.My experiment turned to muti-barreled pipette to compare the variation of neuron firing before and after sodium hydrosulfide was subjected by electrophosis.After that, the brain of rats was taken for histological study to verify the existence of CBS in RVLM so as to shed light on the corollary that endogenous hydrogen sulfide may play a role in this area.Although there are arguments on central oxygen sensor,pre-sympathetic neurons in RVLM own some properties of oxygen sensitivity.Nucleus of Solitary tract(NTS), which receive fibers originated from respiratory center,monosynapthitcally innervates RVLM and assign presympathetic neurons respiratory rhythm.Even on the animals whose carotid body is denervated,hypoxia is still able to increase the activity of sympathetic system and phrenic nerve.Another evident is that C1 cells of rats might have hypoxia sensitivity because they express higher lever of hypoxia induced factor 1-α(HIF1-α) and tyrosine hydroxylase,even after their carotid bodies have been denervated.In addition Local hypoxia mimicked by sodium cyanide will directly increase the firing frequency of some neurons in RVLM domain.The recent research in our laboratory indicated that H2S might involve in the hypoxia reaction of carotid body since H2S increases frequency and shortens the latent period of carotid body firing.It inspires me to make a rudiment study on the effect of trio sulfide when it is subjected focally on presympathetic neurons.Objective1.Observe the change in firing frequency of presympathetic neurons when they are subjected sodium hyposulfite the donor of hydrogen sulfide,by micro electrophoresis. And observe the existence of CBS in RVLM. 2.Observe the change in firing frequency of presympathetic neurons when they are subjected sodium trio sulfide,which is used to mimic local hypoxia.Materials and methods1.Experiments were performed on SD rats.Take tracheal intubation and then cannulate in right arterial femoralis and vena femoralis on both sides after the rat was anesthetized by sodium pentobarbital(1%,50mg/kg).Manage artificial ventilation and continues infusion of vecuronim,a muscle relaxant after arterial blood pressure and ECG were established.The rat's head was fixed on stereo director by two metal nail in its ears.Expose medulla oblongata after get rid of occipital bone and most part of cerebellum.Anesthesia was maintained by pentobarbital bolus 0.2 mini liter per 30 minutes.2.Make a muti-barreled pipette,and fill it with right solutions.After fix it on stereo director,connect it with micro-electrode amplifier and electropherosis.Move the pipette right upon the surface of medullar under which is RVLM and pierce it into the brain.3.The record of neurons firing was began when the pipette reach the depth of 2.3 mm from and ended at the depth 3.7 mm,containing the border of RVLM domain.Each firing was verified by baroreflection led by a bolus of phenylephrine.The ones whose firing was inhibited were classified into experiment group,while the ones which is lack of response to the elevated pressure were in controlled group.Both groups were subject sodium hydrosulfide and sodium trio sulfide during the experiments.The variations of firing before and after subjection were compared.Results1.In the study on the influence of sodium hyposulfite on the cells in RVLM,we investigated 40 samples,among which there are 20 presympathetic neurons and 20 nonpresympathetic neurons.The currents for electrophoresis were -60nA,-90nA and-120nA and last for 30 seconds.20,21and 19 neurons were excited,including 19,20 and 18 presympathetic neurons respectively. 2.Sodium hydrosulfide exerts exciting-inhibiting biphasic effect on presympathetic neurons,each time of electrophoresis,9,11,and 13 neurons out of 20 presympathetic neurons show significant biphasic effect under different current.3.11 out of 19 non-presympathetic neurons were inhibited and the other 8 cells had no explicit reaction towards sodium hydrosulfide.None of these cells was excited by sodium hydrosulfide.4.In histological study,the specific antibody of CBS was subjected to slice of RVLM as the primary antibody,The result shows that the areas adjacent to neurons are stained by ABC.Conclusion1.Sodium hydrosulfide exerts biphasic effect on presympathetic neurons2.Adjacent areas of neurons in RVLM express CBS,which indicates the probability of the existence of endogenous hydrogen sulfide in this area.3.Sodium trio sulfide is ability to excite presympathetic neurons.And from that it is probably implies that presympathetic neurons owns independent oxygen sensitivity.
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