大鼠中缝背核NOS阳性神经元的传入联系及电针对其NOS阳性神经元表达的影响
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摘要
中缝背核是中枢5-羟色胺(5-HT)能神经元的主要聚集部位之一，参与睡眠与觉醒、内分泌活动、情感活动、体温及饮食等多种生理功能的调节，并在疼痛和针刺镇痛过程中起重要作用。研究资料表明，一氧化氮(NO)也是存在于神经系统的一种递质，参与中枢对伤害性信息的处理和调控，且中缝背核内有较密集的一氧化氮合酶(NOS)阳性神经元胞体和纤维分布，那么中缝背核内NOS阳性神经元来源于何处，相关神经递质的活性，如NO等在针刺镇痛过程中有无节律性变化，不同时程、频率、时辰电针对其表达有何影响，尚未见报道，因此值得深入研究。为此我们采用辣根过氧化物酶(HRP)逆行追踪法与还原型辅酶Ⅱ黄递酶(NADPH-d)组织化学方法相结合的技术，研究了大鼠中缝背核NOS阳性神经元的传入联系，实验结果表明，中缝背核接受蓝斑、黑质、隔核和海马等部位的NOS阳性神经元的投射；用NADPH-d组化法、计数法及计算机图像分析相结合的技术，研究了不同时程、频率、时辰电针对中缝背核NOS阳性神经元表达的影响，结果显示，用低频(2Hz)电针大鼠一侧“足三里”穴5、15、30、60分钟，中缝背核内NOS阳性神经元的表达在电针5分钟时，与对照组相比无显著差异(P＞0.05)；电针15分钟后，中缝背核内NOS阳性神经元表达上调(P＜0.05)，但15分钟、30分钟、60分钟各时程组相比无显著差异(P＞0.05)。用低频(2Hz)和高频(128 Hz)电针大鼠一侧“足三里”穴30分钟，中缝背核内NOS阳性神经元的数量较对照组增加(P＜0.05)，其数量与频率呈正相关。于不同时辰(5：00、11：00、17：00、23：00)用低频(2Hz)电针大鼠一侧“足三里”穴30分钟，中缝背核的NOS的表达以5：00上调最为显著(P＜0.05)，其余各时辰组间无显著差异(P＞0.05)。本实验旨在从细胞和分子水平研究DR与NO两者在疼痛调节和针刺镇痛过程中所起的作用和相
安徽医科大学硕士学位论文
互关系，为中缝背核的机能研究及阐明N0的生理机制提供进一步的形态学依据，
为临床针灸提供一些参考资料。
DR is one of the most collective part of serotonin(5-HT) in the nerve center. It takes part in the regulation of sleep and awakening, incretion,emotion,temperture,feeding and so on, and it plays an important role in pain and electroacupuncture(EA) analgesia. Data show that nitric oxide(NO) is another transmitter in nervous system.In the center it regulates harmful informations.There are collective NOS positive neuron bodies and fibers in DR. Where do they come from? Are there diurnal rhythm change during EA? What about the effects of different duration, time and frequency on the expression of NOS positive neurons in DR? There are no data, so we use nicotinamide adenosine dinucleotide phosphate diaphorase (NADPH-d) combing with horseradish peroxidase(HRP) retrograde tracing to evaluate the afferent connections of NOS positive neurons of DR. Results shows that there are NOS positive neurons in locus ceruleus, substantia nigro, septal nucleus and hippocampus projecting to DR. The methods of NADPH-d, counting an
d computer image manipulation technology are adopted to study the effects of different duration, time and frequency on the expression of NOS positive neurons in DR. Data shows that after low frequency(2HZ) EA on "Zusanli" point of one side for 5 min, 15min, 30min, 60min, there are no difference between control group and EA 5 min group, while the NOS positive neurons increases after EA 15 min. There are no obvious difference among EA 15min, 30min and 60min groups (P>0.05). After low (2HZ) and high (128HZ) frequency EA 30 min, the number of NOS positive neurons is increased than that of control group (P<0.05) ,and there is
positive correlation between number and frequency. After low frequency(2HZ) EA at different time(5:00,l 1:00,17:00,23:00) about 30 min, the expression NOS positive neurons in DR increase more obviously at 5:00, the rest groups are no obvious difference. Aim of the test is to research the roles and relationships of DR and NO during regulating pain and EA analgesia from the level of the cell and molecule , and to provide further morphological data for studing the functions of DR, understanding the physiological funtion of NO, and to provide some reference for clinical acupuncture.
安徽医科大学硕士学位论文
互关系，为中缝背核的机能研究及阐明N0的生理机制提供进一步的形态学依据，
为临床针灸提供一些参考资料。
DR is one of the most collective part of serotonin(5-HT) in the nerve center. It takes part in the regulation of sleep and awakening, incretion,emotion,temperture,feeding and so on, and it plays an important role in pain and electroacupuncture(EA) analgesia. Data show that nitric oxide(NO) is another transmitter in nervous system.In the center it regulates harmful informations.There are collective NOS positive neuron bodies and fibers in DR. Where do they come from? Are there diurnal rhythm change during EA? What about the effects of different duration, time and frequency on the expression of NOS positive neurons in DR? There are no data, so we use nicotinamide adenosine dinucleotide phosphate diaphorase (NADPH-d) combing with horseradish peroxidase(HRP) retrograde tracing to evaluate the afferent connections of NOS positive neurons of DR. Results shows that there are NOS positive neurons in locus ceruleus, substantia nigro, septal nucleus and hippocampus projecting to DR. The methods of NADPH-d, counting an
d computer image manipulation technology are adopted to study the effects of different duration, time and frequency on the expression of NOS positive neurons in DR. Data shows that after low frequency(2HZ) EA on "Zusanli" point of one side for 5 min, 15min, 30min, 60min, there are no difference between control group and EA 5 min group, while the NOS positive neurons increases after EA 15 min. There are no obvious difference among EA 15min, 30min and 60min groups (P>0.05). After low (2HZ) and high (128HZ) frequency EA 30 min, the number of NOS positive neurons is increased than that of control group (P<0.05) ,and there is
positive correlation between number and frequency. After low frequency(2HZ) EA at different time(5:00,l 1:00,17:00,23:00) about 30 min, the expression NOS positive neurons in DR increase more obviously at 5:00, the rest groups are no obvious difference. Aim of the test is to research the roles and relationships of DR and NO during regulating pain and EA analgesia from the level of the cell and molecule , and to provide further morphological data for studing the functions of DR, understanding the physiological funtion of NO, and to provide some reference for clinical acupuncture.
引文
1. Taber E., A. Brodal and F. Walberg. The raphe nuclei of the brain stem in the cat.Ⅰ. Normal topography and cytoarchitecture and general discussion. J Comp Neurol,1960;114:161～187
2．刘少雄，许鹿希．大鼠脑干 5-羟色胺神经元的分布-免疫细胞化学研究和定量分析．神经解剖学杂志，1987；3(2)：169
3. Fessler RG. Evidence that the medial and dorsal raphe nuclei mediate serotonergically induced increases in prolaction release from the pituitary. Brain Res, 1984; 299:231
4. Werner J, et al. The Significance of nucleus raphe dorsalis and centralis for thermoafferent signal transmission to the preoptic area of the rat. Exp Brain Res, 1985; 59:543
5. Wang QP, Guan JL,Nakai Y. Distribution and synaptic relations of NOS neurons in the dorsal raphe nucleus: a comparison to 5-HT neurons[J]. Brain Res,1995; 37(2): 177～187
6．韩济生．针刺镇痛原理研究．针刺研究，1984；3(4)：231
7. Grahn RE, Watkins LR, Maier SF. Impaired escape performance and enhanced
conditioned fear in rats following exposure to an uncontrollable stressor are mediated by glutamate and nitric oxide in the dorsal raphe nucleus[J]. Behav Brain Res, 2000; 112(1-2): 33～41
8. Burlet S, Lerger L, Cespuglio R. Nitric oxide and sleep in the rat: a puzzling relationship[J]. Neuroscience, 1999; 92(2): 627～639
9. Meller ST, Gebhart GF. Nitric oxide(NO) and nociceptive processing in the spinal cord. Pain, 1993;52:127
10. Haley JE, Dickenson AH, Schachter M. Electrophysiological evidence for a role of nitric oxide in prolonged chemical nociception in rat. Neuropharmacology, 1992;31:251
11. Gonzalez A, Munoz M, Marin O, et al. Nitric oxide synthase in the brain of a urodele amphibian(Pleurodels Waltt) and its relations to catecholaminergic structures. Brain Res, 1996 Jul; 727(1-2): 49
12. Kondo T, Inokuchi T, Ohtak, et al. Distribution, chemical coding and origin of nitric oxide synthase-containg nerve fibers in the guinea pig nasal mucosa. J Auton Nerve syst ,2000 Apr;80(1-2):71
13．田美玲，凌树才．一氧化氮合酶神经元在大鼠中脑导水管周灰质和中缝核簇内的定位研究．解剖学杂志，1999：22(1)：6～8
14．孙贤奎，梅俊．一氧化氮合成酶阳性神经元在大鼠脑干的分布及其衰老的变化．解剖学杂志，1998：21(4)：308～310
15．余福林，单红英，董新文．大鼠部分脑区内一氧化氮合酶与经典递质的共存．解剖学报，1996：27(2)：153～157
16．黄登凯，周敬修，李宽严，等．大白鼠中缝背核的纤维联系．解剖学报，1984；15(2)：156
17．孙贤奎，楚宪襄，方智慧，等．大鼠中缝背核三种递质传入纤维的脑干起源—免疫细胞化学和酶组织化学双重标记法．解剖学杂志，1996；19(1)：35～37
18．祝善乐，李光千，范玉华，等．大鼠肺内NOS阳性神经极其来源的研究．中国
组织化学与细胞化学杂志，1996；5(1)：22
19．熊克仁，杨解人，郑培敏．大鼠隔区向缰核的一氧化氮合酶阳性神经元的投射．针刺研究，1998；23(1)：21～23
20．熊克仁，李怀斌，郑培敏．大鼠海马接受来自隔区NOS阳性神经纤维．针刺研究，1999；24(3)：184～186
21．王生福，姜平，童鑫康，等．大鼠中缝背核一氧化氮合酶神经元投射分布于大脑皮质一氧化氮合酶神经元．江苏大学学报 (医学版)，2002；12(5)：433～435
22．肖明，丁炯，吴凌霞，等．大鼠中缝背核内一氧化氮合酶神经元向中央杏仁核的投射．南京医科大学学报，2002；22(2)：132～134
23．熊克仁，汪桐．电针对大鼠脊髓和脊神经节一氧化氮表达的影响．针刺研究，1998：23(1)：41～43
24．孔祥玉，赵淑敏，谢宏林，等．针刺时间延长对颗粒小泡 (LVG)与突触部位的影响．解剖学杂志，2000；23(增刊)：222
25．张英．不同灸治时程对红细胞免疫功能影响的比较．中国针灸，2000；20(10)：613
26．李峰，笪翠娣，周敬修．鼠尾伤害性刺激、针刺和针刺镇痛后弓状核—导水管周围灰质—延髓头端腹内侧区的NOS阳性神经元的变化．针刺研究，1997；22(1-2)：55
27．顾耀敏，陈以慈，叶鹿鸣．钨酸钠做为稳定剂的新的高灵敏的HRP-TMB法—Ⅰ光镜研究．神经解剖学杂志，1990；1：121
28. Konig J, Klippel R. The rat brain Baltimore: The Williams and wilkins company, 1963
29．王平宇．大白鼠中枢神经解剖学基础．北京：人民卫生出版社，1986
30. Tong XK, Hamel E. Regional cholinergic denervation of cortical microvessels and nitric oxide synthase containing neurons in Alzheimer s disease [J]. Neuroscience, 1999;92(3): 163～175
31．周兰仙，等．P物质和亮-脑啡肽免疫反应样神经元在鼠脑中缝核的分布．解剖学报，1988：19(3)：256
32. Mocore RY, Halaris AE, Jones BE. Serotonin neurons of midbrain raphe: Ascending projections. J Comp Neurol, 1978;180:417.
33．李云庆．中脑边缘镇痛环路．中国疼痛医学杂志，2000；6(2)：104～108
34. Li YQ, Takad M, Shinonaga Y, et al. Direct projections from the midbrain periaqueductal gray and the dorsal raphe nucleus to the trigeminal sensory complex in the rat. Neuroscience, 1993; 54:431
35．晋志高，范天生．猫前脑和脑干向中缝背核的纤维投射．解剖学报，1988；19(1)：30
36. Zhu H,Zhou W. Morphine induces synchronous oscillatory discharges in the rat locus coeruleus. J Neurosci, 2001 Nov 1;21(21): RC179
37. Xu ZQ ,De Vente J, Steinbusch H, et al. The NO-cGMP pathway in the rat locus coeruleus: electrophysiological, immtmohistochemical and in situ hybridization studies. Eur J Neurosci, 1998; 10:3508～3516
38．沈伟哉，郭国庆，邢旭光，等．大鼠脑干神经元型一氧化氮合酶免疫阳性神经元的分布．解剖学研究，2002；24(2)：138～141
39. Robinson SE. Cardiovascular effects of a-adrenergic agents in the dorsal raphe nucleus of rat. Brain Res, 1984; 295:249
40. Kaehler ST, Sinner C, Philippu A. Release of catecholamines in the locus coeruleus of freely moving and anaesthetized normotensive and spontaneously hypertensive rats: effects of cardiovascular changes and tail pinch. Naunyn Sehmiedebergs Arch Pharmacol, 2000; 361:433～439
41. Sawamura S, Kingery WS, Davies ME et al. Antinociceptive action of nitrous oxide is mediated by stimulation of noradrenergic neurons in the brainstem and activation of [alpha]2B adrenoceptors. J Neurosci, 2000; 20:9242～9251
42．周国庆，陈光辉，张隽，等．帕金森病大鼠模型诱导型一氧化氮合酶基因表达
2．刘少雄，许鹿希．大鼠脑干 5-羟色胺神经元的分布-免疫细胞化学研究和定量分析．神经解剖学杂志，1987；3(2)：169
3. Fessler RG. Evidence that the medial and dorsal raphe nuclei mediate serotonergically induced increases in prolaction release from the pituitary. Brain Res, 1984; 299:231
4. Werner J, et al. The Significance of nucleus raphe dorsalis and centralis for thermoafferent signal transmission to the preoptic area of the rat. Exp Brain Res, 1985; 59:543
5. Wang QP, Guan JL,Nakai Y. Distribution and synaptic relations of NOS neurons in the dorsal raphe nucleus: a comparison to 5-HT neurons[J]. Brain Res,1995; 37(2): 177～187
6．韩济生．针刺镇痛原理研究．针刺研究，1984；3(4)：231
7. Grahn RE, Watkins LR, Maier SF. Impaired escape performance and enhanced
conditioned fear in rats following exposure to an uncontrollable stressor are mediated by glutamate and nitric oxide in the dorsal raphe nucleus[J]. Behav Brain Res, 2000; 112(1-2): 33～41
8. Burlet S, Lerger L, Cespuglio R. Nitric oxide and sleep in the rat: a puzzling relationship[J]. Neuroscience, 1999; 92(2): 627～639
9. Meller ST, Gebhart GF. Nitric oxide(NO) and nociceptive processing in the spinal cord. Pain, 1993;52:127
10. Haley JE, Dickenson AH, Schachter M. Electrophysiological evidence for a role of nitric oxide in prolonged chemical nociception in rat. Neuropharmacology, 1992;31:251
11. Gonzalez A, Munoz M, Marin O, et al. Nitric oxide synthase in the brain of a urodele amphibian(Pleurodels Waltt) and its relations to catecholaminergic structures. Brain Res, 1996 Jul; 727(1-2): 49
12. Kondo T, Inokuchi T, Ohtak, et al. Distribution, chemical coding and origin of nitric oxide synthase-containg nerve fibers in the guinea pig nasal mucosa. J Auton Nerve syst ,2000 Apr;80(1-2):71
13．田美玲，凌树才．一氧化氮合酶神经元在大鼠中脑导水管周灰质和中缝核簇内的定位研究．解剖学杂志，1999：22(1)：6～8
14．孙贤奎，梅俊．一氧化氮合成酶阳性神经元在大鼠脑干的分布及其衰老的变化．解剖学杂志，1998：21(4)：308～310
15．余福林，单红英，董新文．大鼠部分脑区内一氧化氮合酶与经典递质的共存．解剖学报，1996：27(2)：153～157
16．黄登凯，周敬修，李宽严，等．大白鼠中缝背核的纤维联系．解剖学报，1984；15(2)：156
17．孙贤奎，楚宪襄，方智慧，等．大鼠中缝背核三种递质传入纤维的脑干起源—免疫细胞化学和酶组织化学双重标记法．解剖学杂志，1996；19(1)：35～37
18．祝善乐，李光千，范玉华，等．大鼠肺内NOS阳性神经极其来源的研究．中国
组织化学与细胞化学杂志，1996；5(1)：22
19．熊克仁，杨解人，郑培敏．大鼠隔区向缰核的一氧化氮合酶阳性神经元的投射．针刺研究，1998；23(1)：21～23
20．熊克仁，李怀斌，郑培敏．大鼠海马接受来自隔区NOS阳性神经纤维．针刺研究，1999；24(3)：184～186
21．王生福，姜平，童鑫康，等．大鼠中缝背核一氧化氮合酶神经元投射分布于大脑皮质一氧化氮合酶神经元．江苏大学学报 (医学版)，2002；12(5)：433～435
22．肖明，丁炯，吴凌霞，等．大鼠中缝背核内一氧化氮合酶神经元向中央杏仁核的投射．南京医科大学学报，2002；22(2)：132～134
23．熊克仁，汪桐．电针对大鼠脊髓和脊神经节一氧化氮表达的影响．针刺研究，1998：23(1)：41～43
24．孔祥玉，赵淑敏，谢宏林，等．针刺时间延长对颗粒小泡 (LVG)与突触部位的影响．解剖学杂志，2000；23(增刊)：222
25．张英．不同灸治时程对红细胞免疫功能影响的比较．中国针灸，2000；20(10)：613
26．李峰，笪翠娣，周敬修．鼠尾伤害性刺激、针刺和针刺镇痛后弓状核—导水管周围灰质—延髓头端腹内侧区的NOS阳性神经元的变化．针刺研究，1997；22(1-2)：55
27．顾耀敏，陈以慈，叶鹿鸣．钨酸钠做为稳定剂的新的高灵敏的HRP-TMB法—Ⅰ光镜研究．神经解剖学杂志，1990；1：121
28. Konig J, Klippel R. The rat brain Baltimore: The Williams and wilkins company, 1963
29．王平宇．大白鼠中枢神经解剖学基础．北京：人民卫生出版社，1986
30. Tong XK, Hamel E. Regional cholinergic denervation of cortical microvessels and nitric oxide synthase containing neurons in Alzheimer s disease [J]. Neuroscience, 1999;92(3): 163～175
31．周兰仙，等．P物质和亮-脑啡肽免疫反应样神经元在鼠脑中缝核的分布．解剖学报，1988：19(3)：256
32. Mocore RY, Halaris AE, Jones BE. Serotonin neurons of midbrain raphe: Ascending projections. J Comp Neurol, 1978;180:417.
33．李云庆．中脑边缘镇痛环路．中国疼痛医学杂志，2000；6(2)：104～108
34. Li YQ, Takad M, Shinonaga Y, et al. Direct projections from the midbrain periaqueductal gray and the dorsal raphe nucleus to the trigeminal sensory complex in the rat. Neuroscience, 1993; 54:431
35．晋志高，范天生．猫前脑和脑干向中缝背核的纤维投射．解剖学报，1988；19(1)：30
36. Zhu H,Zhou W. Morphine induces synchronous oscillatory discharges in the rat locus coeruleus. J Neurosci, 2001 Nov 1;21(21): RC179
37. Xu ZQ ,De Vente J, Steinbusch H, et al. The NO-cGMP pathway in the rat locus coeruleus: electrophysiological, immtmohistochemical and in situ hybridization studies. Eur J Neurosci, 1998; 10:3508～3516
38．沈伟哉，郭国庆，邢旭光，等．大鼠脑干神经元型一氧化氮合酶免疫阳性神经元的分布．解剖学研究，2002；24(2)：138～141
39. Robinson SE. Cardiovascular effects of a-adrenergic agents in the dorsal raphe nucleus of rat. Brain Res, 1984; 295:249
40. Kaehler ST, Sinner C, Philippu A. Release of catecholamines in the locus coeruleus of freely moving and anaesthetized normotensive and spontaneously hypertensive rats: effects of cardiovascular changes and tail pinch. Naunyn Sehmiedebergs Arch Pharmacol, 2000; 361:433～439
41. Sawamura S, Kingery WS, Davies ME et al. Antinociceptive action of nitrous oxide is mediated by stimulation of noradrenergic neurons in the brainstem and activation of [alpha]2B adrenoceptors. J Neurosci, 2000; 20:9242～9251
42．周国庆，陈光辉，张隽，等．帕金森病大鼠模型诱导型一氧化氮合酶基因表达