【作者】 李自强；

【导师】 吴中海；

【作者基本信息】 中国人民解放军第一军医大学 ， 神经生理学， 2003， 硕士

【摘要】 目的 1)应用新生大鼠离体延髓脑片标本，记录与之相连的舌下神经呼吸节律性放电(respiratory rhythmical discharge activity，RRDA)，在灌流液中分别给予不同浓度的NO(nitric oxide，NO)供体硝普钠(sodium nitropmsside，SNP)，NO合成前体L-精氨酸(L-Arginine，L-Arg)以及神经元型一氧化氮合酶(neuronal nitric oxide synthase，nNOS)特异性抑制剂7-nitro indazole (7-NI)，观察其对RRDA的影响，从而探讨NO对节律性呼吸的产生和调节中的作用；2)在新生大鼠离体延髓脑片标本上，同步记录舌下神经根和面神经后核内侧区(the medial area of retrofacialis nucleus，mNRF)中的吸气神经元的放电活动，分别给以NO的合成前体、供体以及nNOS的特异性抑制剂，进一步探讨NO对节律性呼吸产生影响的可能机制。 方法 选用新生SD大鼠(0～3d)，雌雄不拘。1)参照并改良Suzue的方法制作离体延髓脑片标本，主要包含面神经后核内侧区，前包钦格氏复合体(Pre-B(?)tzinger Complex)、腹侧呼吸组以及背侧呼吸组的一部分，保留舌下神经根。用玻璃吸附电极记录舌下神经放电作为呼吸活动的指标，主要的呼吸参数包括呼吸周期(respiratory cycle，RC)、吸气时程(inspiratory time，TI)、呼气时程(expiratory time，TE)以及放电的积分幅度(integral amplitude，IA)，通过灌流液给药，观察不同浓度的不同药物对神经根放电活动的影响；2)在面神经后核内侧区(mNRF)同步记录吸气神经元单位的放电活动，灌流给予上述不同药物药物，观察其对吸气神经元放电的影响。 结果 1在记录神经根的实验部分：1)灌流给予不同浓度的NO合成前体L-Arg(从500μmol/L到2mmol/L)，与给药前对照相比，舌下神经根的各项放电指标均未出现显著性变化;2)灌流给予不同浓度的NO供体sNP(浓度100脚。UL~2~1几)，不论给予上述任何浓度的SNP，舌下神经根放电指标与对照相比都未出现明显变化;3)灌流给予不同浓度的nNOs的特异性抑制剂7一I(500娜ol/L~2~ol几)后，Rc、TE无显著性变化，而随着给药时间的逐渐延长，TI和IA均同时出现逐渐的下降的趋势:500娜。比7一I实验组，正常MKS液灌流时TI为1.39士0.055、I^为1387.00士65.16林V·ms，给药后第smin时Tl为1.01士0 .055、I^为1274.00士55.82”V·ms(p<0.05)，第10min时TI为0.89士0.055、IA为1218.00士55.34衅·ms(p<0 .05);lmmo比的7一I实验组，正常对照组Tl为1.01士0.185、IA为1642.83士226.90四.ms，给药后Slnin，Tl为0.87士 0.145、认为1528.43士189.02四·ms(P<0 .05)，10rliln时，Tl为0 .37士0.125、IA为1278.83士180.92四.ms(P<0 .05);Znnno讥7一I实验组，正常对照的TI为1.17士0.065、IA为1356.00士65.16四·ms，给药后smin时，Tl为0.87士0.055、IA为1190.00士57.82衅.ms(P<0 .05)，给药10min时，Tl为0.32士0.035、IA为966.00士58.34四.ms(’I’<0 .05)。7一I的浓度和这种抑制作用还存在量效关系，10min时，给予500洲mo比Tl下降约36%，IA下降12%，Inuno比时TI下降63%，IA下降22%，2~泥时Tl下降73%，IA下降28%;4)单纯给予相应浓度的7一I溶剂二甲亚矾(d如ethyl sulfoxide，DMSO)后，舌下神经根放电活动无显著性变化。 2同步记录舌下神经根和mNRF区吸气神经元放电活动的实验:l)给药前，正常MKS灌流时，吸气神经元放电的平均频率为25.39士5.04次HZ，放电时程为0.5811士0.02975;2)先后分别给予L一A飞、SNP之后，吸气神经元放电的平均频率分别为26.79士5.llHz和26.43土5.02Hz，放电时程分别为0.5733士0.02535和0.5628士0.02655，与对照相比平均频率和放电时程并无显著性变化(卜0.05);3)给予nNOS特异性抑制剂7一I之后，吸气神经元的放电时程为0.1894士0.02475，平均频率为17.58士4.78HZ，和对照相比均显著性下降(P<0 .05)，分别下降67.4%和30.8%:4)灌流给予上述三种试剂后，TE均未发生明显变化。 结论1.NO对节律性呼吸中吸气活动的抑制作用参与了吸气中止机制;2.NO对呼吸强度和呼吸频率均有调节作用，但对前者的作用更为重要;3.在NO对基本节律性呼吸的调节过程中，主要由神经元合成并释放的内源性NO就可能满足其对节律性呼吸的调节功能的需要，而外源性NO，则可能由于对其需要量不大，或由于其半衰期过短，所以在呼吸调节中的作用不大;4.NO对mNRF中吸气神经元的放电时程和放电平均频率有明显的抑制作用，提示NO对吸气中止机制的影响，可能是通过对mN盯的吸气神经元的作用而实现的。更多还原

【Abstract】 Nitric oxide is involved in the modulation of the basic rhythmical respiration in vitro brainstem slice from neonatalratObject1. This experiment was expected to test whether NO (nitric oxide) exerted significant effects on basic rhythmical respiration.2. To investigate the role of NO in the basic rhythmical respiration generation and modulation by exploring the effects of substrate N donor of NO and inhibitor of nNOS (neuronal nitric oxide synthase) on the discharge activity of inspiratory neurons in mNRFMethods:Experiments were performed on in vitro brainstem slice preparations isolated from neonatal rats (0-3 d). These preparations include the medial region of the nucleus retrofacialis (mNRF); a part of pre-Botinger Complex, ventral respiratory group (VRG) and dorsal respiratory group (DRG).l)Respiratory-related burst activities were recorded from hypoglossal nerve rootlets before and during perfusion of the slice preparation with L-Arg (L-Arginine), SNP(sodium nhroprusside) or 7-NI (7-nitro indazole, an inhibitor of neuronal nitric oxide synthase).2)As monitoring the respiratory-related burst activities of hypoglossal nerve rootlets, we recorded the discharge of inspiratory neurons in the mNRF by inserting the glass microeletrode into the mNRF of the slice. These inspiratory neurons were determined according to the temporal relationship between the neuronal discharge and the hypoglossal nerve activity. Drugswere administered by bath application and their effects on the inspiratoryneuronal activity were investigated.Results:1. Results from experiments on recording respiratory-related burst activities of hypoglossal nerve rootlets. 1) After perfused with different concentrations of L-Arg (500@mol/L~2mmol/L) and SNP (100@mol/L~2mmol/L) , there was no significant change in RRDA; 2) 7-NI, an inhibitor of nNOS, had no effect on the TE, RC,but it decreased the integral amplitude of burst (IA) and inspiratory time (TI), and this kind of inhibition is dose-dependent and time-dependent: during the control perfusion, TI(inspiratory time) is 1.39 ± 0.05s, IA (integral amplitude) is 1387.00 + 65.16@V.ms , at 5min after application of 500@mol/L 7-NI, TI is 1.01±0.05s, IA is 1274.00 ±55.82@Vms (P<0.05), and at lOmin, TI is 0.89±0.05s, IA is 1218.00+55.34uV’ms (P<0.05) . In 1mmol/L 7-NI group, before given the drug, TI is 1.01 ± 0.18s, IA is 1642.83±226.90@V.ms; at 5min after given the drug, TI is 0.87±0.14s, IA is 1528.43±189.02@V.ms (P<0.05); at lOmin, TI is 0.37 + 0.12s, IA is 1278.83+ 180.92uV-ms (PO.05); In 2mmol/L 7-NI group, TI is 1.17+0.06s, IA is 1356.00+65.16uV-ms when perfusion with MKS; at 5min, TI is 0.87±0.05, IA is 1190.00±57.82uV-ms (PO.05); at lOmin, TI is 0.32 +0.03s, IA is 966.00+ 58.34uV’ms (PO.05). As shown above, at lOmin after addition of different concentrations of 7-NI, TI decreased 36%, IA decrease 12% when the concentration of 7-NI is 500umol/L; when the concentration of 7-NI is Immol/L, TI decrease 63%, IA decrease 22%; at 2mmol/L, TI decrease 73 %, IA decrease 28 %. 3) After only given the same concentration of DMSO (dimethyl sulfoxide, DMSO) as dissolving the 7-NI, there is no significant difference between the MKS group and experimental group.2. Result from experiments on recording of discharge of inspiratory neurons in the mNRF: 1) In the MKS group, TI is 0.5811 ± 0.0297s , Fn (average neuronal discharge frequency) is 25.39 ± 5.041Hz; 2) Afergiven L-Arg, SNP respectively, Fn is 26.79 ± 5.11 Hz and 26.43 ± 5.02Hz , TI is 0.5733 ± 0.0253s and 0.5628 ± 0.0265s , there is no significant change compared with the control group (P>0.05) ; 3) After application of 7-NI, TI is 0.1894 ± 0.0247s, Fn is 17.58±4.783Hz, they decrease 67.4% and 30.8% respectively.Conlusion1. NO may take part in the IOS (inspiratory off-switching mechanism because of its regulation to the inspiration.2. NO can regulate the amplitude and frequency of respiration, but the regulation on amplitude of respiratory bursts of NO is more powerf更多还原