氦氖激光对缺血缺氧新生大鼠脑超微结构及ChAT、BDNF表达的影响
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摘要
背景与目的：
缺血缺氧性脑损伤(Hypoxic-ischemic brain damage,HIBD)是威胁新生儿生命的严重疾病，具有很高的死亡率，是引起智能低下和脑性瘫痪的主要原因之一。目前，国内外学者对缺血缺氧性脑损伤的治疗作了大量的研究工作，取得了一定进展，但临床上尚缺乏较为有效的治疗措施，故寻找更有效的防治方法，成了围产医学及神经科学工作者一项迫切的任务。氦氖激光穴位照射是一种非侵入性治疗，具有柔和、无痛、无菌的特点，同时兼有独特的心理和精神康复作用，是具有应用前景的治疗手段。氦氖激光作为一种低能量激光，由于其特有的生物刺激效应已被应用于临床，但是关于其能否促进新生儿脑损伤后脑的修复和发育及其作用机制鲜有报道。因此本实验采用国际公认的新生大鼠缺血缺氧模型，选用督脉经穴—百会和大椎，观察氦氖激光穴位照射对脑缺血缺氧新生大鼠脑组织超微结构的改变及对脑神经元胆碱乙酰基转移酶(Choline Acetyltrans ferase,ChAT)，脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)表达的影响，旨在探讨氦氖激光穴位照射对新生儿缺血缺氧性脑病的作用及其机制，并为临床寻找安全高效的治疗方法提供实验依据。
方法：
62只健康7日龄Wistar大鼠，体重12-15g，随机分为3组。Ⅰ 假手术对照组(n=16只)：动物仅接受手术处理，不结扎血管，不缺氧；Ⅱ 缺血缺氧组(n=24只，存活22只)：结扎左侧颈总动脉，然后入8％O_2，92％N_2缺氧2小时；Ⅲ缺血缺氧后氦氖激光穴位照射治疗组(n=22只，存活19只)：缺血缺氧后第2天开始采
郑州大学2003年硕士学位论文
氮氖激光对缺血缺氛新生大鼠脑超微结构及C卜冉T、BDNF表达的影响
用波长632.8nm氦氖激光照射“百会”、“大椎”两穴，功率4mw，光斑直径3mm，
功率密度56.62mv刀c扩，每次10分钟，能量密度33.97J/c扩，每天1次，同一
时间进行，ro天为一疗程，共两疗程，两疗程间隔2天。I和11组大鼠于缺血
缺氧后24h随机各抽取3只，作TTe(2，3，5一tdp阮nyltetrazo川ium。hioride)染色以
判定模型是否成功。上述3组动物常规饲养22天后，每组各再随机抽取3只，
取左侧脑海马组织，制成超薄切片，H-7500型透射电镜观察。其余42只大鼠
常规灌注取材，石蜡包埋、切片、行HE、Nissl和免疫组化染色，光镜下观察，
计数，灰度分析，最后统计学处理。
结果:
1 .HE染色:假手术对照组大脑皮层和海马区神经元细胞层次清楚，排列规则整
齐，细胞形态结构清晰完整;缺血缺氧组大脑皮层和海马区神经元有变性、坏死，
细胞数目减少，结构模糊，氦氖激光穴位照射组大脑皮层和海马区神经元变性坏
死减轻，细胞形态结构较清晰。
2.Nissl染色:假手术对照组大脑皮层和海马区神经元胞浆中布满着深蓝色呈颗粒
状的尼氏体(灰度值:122.9肚12.10)，缺血缺氧组神经元胞浆内尼氏体着色浅淡，
有不同程度的脱失(灰度值:1 88.31月0.43)，与假手术组相比差异有统计学意义
(P<0 .05);氦氖激光穴位照射组神经元胞浆内的尼氏体有所恢复，较缺血缺氧组
明显减轻(灰度值:130.5肚6.16)，两者比较具有统计学意义(P<0 .05)。
3.海马神经元超微结构改变:假手术对照组神经元结构完整，细胞核膜清晰，
胞浆内细胞器丰富，线粒体内崎清晰致密，粗面内质网核糖体丰富，胞浆内微丝
微管排列规则整齐;缺血缺氧组神经元胞体水肿，核膜模糊不清，细胞器明显减
少，线粒体部分峭断裂或溶解消失，粗面内质网扩张，表面核糖体脱落，微丝微
管紊乱;氦氖激光穴位照射组神经元胞体略肿胀，结构较清晰，核膜完整，胞浆
内细胞器较丰富，线粒体峭稍模糊;内质网核糖体较丰富，微丝微管排列较规则
整齐。
4.ChAT免疫组织化学染色:假手术对照组海马结构CAI、CAZ、CA3、齿状回
均有大量Ch灯免疫反应阳性神经元分布，CAI区阳性细胞数平均为29.74士
4.85，缺血缺氧组海马CAI区内Ch灯免疫反应阳性神经元数量减少，平均为
13.%士7.62，与假手术组相比差异有统计学意义俨<。.05)，氦氖激光穴位照射组
郑州大学2003年硕士学位论文
氮氖激光对缺血缺氧新生大鼠脑超微结构及C扮汀、BDN下表达的影响
ChAT免疫反应阳性神经元平均为26.42士6.02，较缺血缺氧组增多，两者比较具有
统计学意义(P<0.05)。
5.BDNF免疫组织化学染色:BDN下免疫阳性神经元主要分布在大脑皮层和海
马CAZ，CA3区，假手术对照组BDN[F免疫阳性细胞数较少(皮层:14，14士6.n;
海马:13 .42土5.56)，而缺血缺氧组BDN’F免疫阳性细胞数明显增加(皮层:24.49
士8.31;海马:21.93巧.12)，与假手术组相比差异有统计学意义(P<0 .05);氦氖
激光穴位照射组BDNF免疫阳性细胞(皮层:3824士8.78;海马:33.02土7.58)
表达较缺血缺氧组显著增加，两者比较具有统计学意义(P<0 .05)。
结论:
1，氖激光穴位照射可以抵抗缺血缺氧造成脑神经元尼氏体的脱失，促进脑神经
元的恢复;
2.氦氖激光穴位照射可以改善缺血缺氧对脑神经元超微结构的损害;
3.氦氖激光穴位照射可使脑神经元ChAI表达增加，提高缺血缺氧后神经元的
功能;
4.氦氖激光穴位照射可使脑
Objective:Hypoxic-ischemic brain damage (HIBD) continues to be a major problem in perinatal medicine because of the high mortality rate and neurological and intellectual impairment in the surviving infants. At present, despite scholars have done lots of investigation in the treatment of HIBD, no effective therapeutic strategies have yet been developed to counteract this condition in clinic, therefore, one of the most urgent tasks for obstetricians and neurologist is to develop therapeutic strategies. He-Ne laser acupoint irradiation is an non-invasive therapy and has soft, painless , sterille features, in addition it has an unique psycho and psychiatric rehabilitation function, so it is a therapeutic tool with applicated prospectAs a low energy laser, He-Ne laser has been applied in clinic because of its unique biological stimulation, but whether it could accelerate brain plerosis and development after neonatal brain injury has not been reported. For this reason our experiment adopted a generally accepted ra
t model with hypoxia-ischemia, selected "BaiHui"point; "DaZhui"point to study the effect of He-Ne laser acupoint irradiation on ultrastructural changes and expression of ChAT and BDNF after hypoxia-ischemia in neonatal rat brain, trying to find out the mechanisms and a safe therapeutic method for clinic.
Methods: Seven-day-old rats (n =62) weighing 12-15g were randomly divided into three groups. I group (n =16), neither ligated nor exposed to hypoxia, served as controls. II group (n =24, 22 survivor) was exposed to hypoxic-ischemic conditions, ligation of the left common carotid artery followed by 2h of hypoxia with 8% oxygen 92% Nitrogen, in group (n =22,19 survivor) was treated with He-Ne laser acupoint irradiation in the next day following hypoxia. (He-Ne laser with wavelength of 632.8 nm, output power of 4mW,light spot diameter of 3mm, power density of 56.62 mW/cm 2and doses of 33.97 J/cm2, the exposure duration 10 min/d, 10 procedures for 1 course, 2 courses with 2d interval). 24h after hypoxia, three rats in group I and II respectively were randomly put to death for TTC staining. 22d later, three rats selected from each group were killed. The left side hippocampus from each rat was dissected out, the specimens were made ultrathin sections prepared for observation by an electron microscope H-7500. The 42 rats were subjected to perfusion-fixation, the left brains were regularly paraffin wax embedded, sectioned and stained by HE staining, Nissl staining, ChAT and BDNF immunohistochemical staining. The neurons were observed, the number of positive neurons counted under microscope and the grey scale values were masured, then data were statistically analysed. Results: (1) HE staining: the structure of neurons in hippocampus and cortex is clear in group I ; There is neuronal degeneration , necrosis and tissue structure clouding in group II; The neuronal denaturalization and necrosis relieve, cellular form and structure are in focus in group III.
(2)Nissl staining : The neuronal cytoplasm is full of dark-blue colored Nissl bodies in group I (grey scale value: 122.90+12.10), a great number of nissl bodies are lost in group II, the grey scale value appears ascending tendency(188.31+10.43), there is obvious difference between I and II (p<0.05). There is a slight loss of Nissl bodies in group III, its grey scale value appears a descending tendency(130.56+6.16), there is marked diference between group II and group III (p<0.05).
(3) The ultrastructural changes in hippocampus: The neuronal structure of hippocampus in group I is integra. nuclear membrane is clear, there are plenty of organells in cytoplasm. Mitochondrial cristae is clear and dense, RER and ribosome
are abundant, microfilament and microtubules array in good order; In group n the ultrastructural damage of neurons result in remarkably cellular edema and clouding nuclear membrane. Organells in cytoplasm decrease, mitochondrial cristae is poor structural integrity. The swollen RER attached fewer ribosomes, microfilament and microtubules arr
缺血缺氧性脑损伤(Hypoxic-ischemic brain damage,HIBD)是威胁新生儿生命的严重疾病，具有很高的死亡率，是引起智能低下和脑性瘫痪的主要原因之一。目前，国内外学者对缺血缺氧性脑损伤的治疗作了大量的研究工作，取得了一定进展，但临床上尚缺乏较为有效的治疗措施，故寻找更有效的防治方法，成了围产医学及神经科学工作者一项迫切的任务。氦氖激光穴位照射是一种非侵入性治疗，具有柔和、无痛、无菌的特点，同时兼有独特的心理和精神康复作用，是具有应用前景的治疗手段。氦氖激光作为一种低能量激光，由于其特有的生物刺激效应已被应用于临床，但是关于其能否促进新生儿脑损伤后脑的修复和发育及其作用机制鲜有报道。因此本实验采用国际公认的新生大鼠缺血缺氧模型，选用督脉经穴—百会和大椎，观察氦氖激光穴位照射对脑缺血缺氧新生大鼠脑组织超微结构的改变及对脑神经元胆碱乙酰基转移酶(Choline Acetyltrans ferase,ChAT)，脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)表达的影响，旨在探讨氦氖激光穴位照射对新生儿缺血缺氧性脑病的作用及其机制，并为临床寻找安全高效的治疗方法提供实验依据。
方法：
62只健康7日龄Wistar大鼠，体重12-15g，随机分为3组。Ⅰ 假手术对照组(n=16只)：动物仅接受手术处理，不结扎血管，不缺氧；Ⅱ 缺血缺氧组(n=24只，存活22只)：结扎左侧颈总动脉，然后入8％O_2，92％N_2缺氧2小时；Ⅲ缺血缺氧后氦氖激光穴位照射治疗组(n=22只，存活19只)：缺血缺氧后第2天开始采
郑州大学2003年硕士学位论文
氮氖激光对缺血缺氛新生大鼠脑超微结构及C卜冉T、BDNF表达的影响
用波长632.8nm氦氖激光照射“百会”、“大椎”两穴，功率4mw，光斑直径3mm，
功率密度56.62mv刀c扩，每次10分钟，能量密度33.97J/c扩，每天1次，同一
时间进行，ro天为一疗程，共两疗程，两疗程间隔2天。I和11组大鼠于缺血
缺氧后24h随机各抽取3只，作TTe(2，3，5一tdp阮nyltetrazo川ium。hioride)染色以
判定模型是否成功。上述3组动物常规饲养22天后，每组各再随机抽取3只，
取左侧脑海马组织，制成超薄切片，H-7500型透射电镜观察。其余42只大鼠
常规灌注取材，石蜡包埋、切片、行HE、Nissl和免疫组化染色，光镜下观察，
计数，灰度分析，最后统计学处理。
结果:
1 .HE染色:假手术对照组大脑皮层和海马区神经元细胞层次清楚，排列规则整
齐，细胞形态结构清晰完整;缺血缺氧组大脑皮层和海马区神经元有变性、坏死，
细胞数目减少，结构模糊，氦氖激光穴位照射组大脑皮层和海马区神经元变性坏
死减轻，细胞形态结构较清晰。
2.Nissl染色:假手术对照组大脑皮层和海马区神经元胞浆中布满着深蓝色呈颗粒
状的尼氏体(灰度值:122.9肚12.10)，缺血缺氧组神经元胞浆内尼氏体着色浅淡，
有不同程度的脱失(灰度值:1 88.31月0.43)，与假手术组相比差异有统计学意义
(P<0 .05);氦氖激光穴位照射组神经元胞浆内的尼氏体有所恢复，较缺血缺氧组
明显减轻(灰度值:130.5肚6.16)，两者比较具有统计学意义(P<0 .05)。
3.海马神经元超微结构改变:假手术对照组神经元结构完整，细胞核膜清晰，
胞浆内细胞器丰富，线粒体内崎清晰致密，粗面内质网核糖体丰富，胞浆内微丝
微管排列规则整齐;缺血缺氧组神经元胞体水肿，核膜模糊不清，细胞器明显减
少，线粒体部分峭断裂或溶解消失，粗面内质网扩张，表面核糖体脱落，微丝微
管紊乱;氦氖激光穴位照射组神经元胞体略肿胀，结构较清晰，核膜完整，胞浆
内细胞器较丰富，线粒体峭稍模糊;内质网核糖体较丰富，微丝微管排列较规则
整齐。
4.ChAT免疫组织化学染色:假手术对照组海马结构CAI、CAZ、CA3、齿状回
均有大量Ch灯免疫反应阳性神经元分布，CAI区阳性细胞数平均为29.74士
4.85，缺血缺氧组海马CAI区内Ch灯免疫反应阳性神经元数量减少，平均为
13.%士7.62，与假手术组相比差异有统计学意义俨<。.05)，氦氖激光穴位照射组
郑州大学2003年硕士学位论文
氮氖激光对缺血缺氧新生大鼠脑超微结构及C扮汀、BDN下表达的影响
ChAT免疫反应阳性神经元平均为26.42士6.02，较缺血缺氧组增多，两者比较具有
统计学意义(P<0.05)。
5.BDNF免疫组织化学染色:BDN下免疫阳性神经元主要分布在大脑皮层和海
马CAZ，CA3区，假手术对照组BDN[F免疫阳性细胞数较少(皮层:14，14士6.n;
海马:13 .42土5.56)，而缺血缺氧组BDN’F免疫阳性细胞数明显增加(皮层:24.49
士8.31;海马:21.93巧.12)，与假手术组相比差异有统计学意义(P<0 .05);氦氖
激光穴位照射组BDNF免疫阳性细胞(皮层:3824士8.78;海马:33.02土7.58)
表达较缺血缺氧组显著增加，两者比较具有统计学意义(P<0 .05)。
结论:
1，氖激光穴位照射可以抵抗缺血缺氧造成脑神经元尼氏体的脱失，促进脑神经
元的恢复;
2.氦氖激光穴位照射可以改善缺血缺氧对脑神经元超微结构的损害;
3.氦氖激光穴位照射可使脑神经元ChAI表达增加，提高缺血缺氧后神经元的
功能;
4.氦氖激光穴位照射可使脑
Objective:Hypoxic-ischemic brain damage (HIBD) continues to be a major problem in perinatal medicine because of the high mortality rate and neurological and intellectual impairment in the surviving infants. At present, despite scholars have done lots of investigation in the treatment of HIBD, no effective therapeutic strategies have yet been developed to counteract this condition in clinic, therefore, one of the most urgent tasks for obstetricians and neurologist is to develop therapeutic strategies. He-Ne laser acupoint irradiation is an non-invasive therapy and has soft, painless , sterille features, in addition it has an unique psycho and psychiatric rehabilitation function, so it is a therapeutic tool with applicated prospectAs a low energy laser, He-Ne laser has been applied in clinic because of its unique biological stimulation, but whether it could accelerate brain plerosis and development after neonatal brain injury has not been reported. For this reason our experiment adopted a generally accepted ra
t model with hypoxia-ischemia, selected "BaiHui"point; "DaZhui"point to study the effect of He-Ne laser acupoint irradiation on ultrastructural changes and expression of ChAT and BDNF after hypoxia-ischemia in neonatal rat brain, trying to find out the mechanisms and a safe therapeutic method for clinic.
Methods: Seven-day-old rats (n =62) weighing 12-15g were randomly divided into three groups. I group (n =16), neither ligated nor exposed to hypoxia, served as controls. II group (n =24, 22 survivor) was exposed to hypoxic-ischemic conditions, ligation of the left common carotid artery followed by 2h of hypoxia with 8% oxygen 92% Nitrogen, in group (n =22,19 survivor) was treated with He-Ne laser acupoint irradiation in the next day following hypoxia. (He-Ne laser with wavelength of 632.8 nm, output power of 4mW,light spot diameter of 3mm, power density of 56.62 mW/cm 2and doses of 33.97 J/cm2, the exposure duration 10 min/d, 10 procedures for 1 course, 2 courses with 2d interval). 24h after hypoxia, three rats in group I and II respectively were randomly put to death for TTC staining. 22d later, three rats selected from each group were killed. The left side hippocampus from each rat was dissected out, the specimens were made ultrathin sections prepared for observation by an electron microscope H-7500. The 42 rats were subjected to perfusion-fixation, the left brains were regularly paraffin wax embedded, sectioned and stained by HE staining, Nissl staining, ChAT and BDNF immunohistochemical staining. The neurons were observed, the number of positive neurons counted under microscope and the grey scale values were masured, then data were statistically analysed. Results: (1) HE staining: the structure of neurons in hippocampus and cortex is clear in group I ; There is neuronal degeneration , necrosis and tissue structure clouding in group II; The neuronal denaturalization and necrosis relieve, cellular form and structure are in focus in group III.
(2)Nissl staining : The neuronal cytoplasm is full of dark-blue colored Nissl bodies in group I (grey scale value: 122.90+12.10), a great number of nissl bodies are lost in group II, the grey scale value appears ascending tendency(188.31+10.43), there is obvious difference between I and II (p<0.05). There is a slight loss of Nissl bodies in group III, its grey scale value appears a descending tendency(130.56+6.16), there is marked diference between group II and group III (p<0.05).
(3) The ultrastructural changes in hippocampus: The neuronal structure of hippocampus in group I is integra. nuclear membrane is clear, there are plenty of organells in cytoplasm. Mitochondrial cristae is clear and dense, RER and ribosome
are abundant, microfilament and microtubules array in good order; In group n the ultrastructural damage of neurons result in remarkably cellular edema and clouding nuclear membrane. Organells in cytoplasm decrease, mitochondrial cristae is poor structural integrity. The swollen RER attached fewer ribosomes, microfilament and microtubules arr
引文
1. Felt BT, Schallert T, Shao J, et al. Early Appearance of Functional Deficits after Neonatal Excitotoxic and Hypoxic-Ischemic Injury: Fragile Recovery after Development and Role of the NMDA Receptor. Dev Neurosci, 2002; 24(5): 418～425
2. Robert C. Vannucci, Robert M. Brucklacher, et al. Intracellular calcium accumulation during the evolution of hypoxic-ischemic brain damage in the immature rat. Dev Brain Res,2001, 26: 117～120
3. Buonocore G, Perrone S, Muraca MC. Free radicals and brain damage in newborns with hypoxic-ischemic lesion. Ann Ist Super Sanita, 2001,37(4): 527～535
4. Bettina M, Frank P, Pierre G.Effects of interleukin-10 on neonatal excitotoxic brain lesions in mice. Dev Brain Res, 2003,141:25～32
5. Mark R. Pulera, Lisa M, Hantao Liu, et al. Apoptosis in a Neonatal Rat Model of Cerebral Hypoxia-Ischemia.Stroke, 1998,29:2622～2630
6．吴明远，石一复．新生儿缺氧缺血性脑损伤的防治进展．中华妇产科杂志，2000，35(10)：630～632
7. Berger R, Garnier Y.Perinatal brain injury. J Perinat Med, 2000, 28:261～285
8．徐国祥，章萍，唐建民等．激光医学．第一版．北京：人民卫生出版社，1998，6：129～134
9. Mirsky N, Krispel Y, Shoshany Y, et al. Promotion of angiogenesis by low energy laser irradiation. Antioxid Redox Signal, 2002, 4(5): 785～790
10. Reddy GK, Stehno-Bittel L, Enwemeka CS. Laser photostimulation accelerates wound healing in diabetic rats. Wound Repair Regen, 2001, 9(3): 248～255
11. Rochkind S, Nissan M, Alon M, et al. Effects of laser irradiation on the spinal cord for the regeneration of crushed peripheral nerve in rats. Lasers Surg Med,2001,28(3): 216～9
12．于晓虹，华仲慰，李玉书．新生鼠半球性脑缺血的实验模型．第一军医大学
学报，1990，10(3)：236～238
13．华兴邦，李辞蓉．大鼠穴位图谱的研制．实验动物与动物实验，1991，(1)：1～5
14．许能贵，许冠荪，钟平等．电针督脉经穴对急性脑缺血大鼠一氧化氮及内皮素的影响．针刺研究，1996，21(3)：18～20
15．刑艳丽，姚凤祯，杜莹莹．头穴针刺次数对中风病人血液流变学的影响．中国针灸，1994，14(4)；37
16．聂卉．电针对中风偏瘫患者体感诱发电位动态观察．中国针灸，1999，19(6)：369
17．马元．浅谈大椎．针灸临床杂志，1994，10(1)：41～42
18．许能贵，周逸平，许冠荪等．电针大椎、百会穴对局灶性脑缺血大鼠脑血流量和自发脑电的影响．中国中医药科技，2001，8(1)：3～5
19．周筠梅．ATP合成酶的结构变化机理和旋转催化．生物化学与生物物理进展，1998，25(1)：9
20．高震．论经穴能量系统的本质及刺激传导机理．中国针灸，2001，21(3)：161～163
21．王冰水，易南，李玲等．不同功率氦氖激光照射与大鼠脊髓降钙素基因相关肽表达的关系．现代康复，2000，4(6)：858～859
22．王冰水，李玲，易南等．氦氖激光照射对大鼠神经损伤后脊髓内生长相关蛋白表达的影响．中华物理医学与康复杂志，2000，22(2)：89～91
23．罗荣辉，刘婉华，罗先润．氦氖激光内关穴照射对老年冠心病患者血流动力学的影响．河南医科大学学报，2001，36(4)：417～418
24．张卫光，夏家骝，陶平等．氦氖激光照射大鼠心前区对血液流变学指标的影响．中华理疗杂志，2000，23(2)：17～19
25．朱长庚．免疫-神经-内分泌网络．解剖学报，1993，24(2)：216～221
26．张红宇，杨桐伟，王广义等．氦氖激光穴位照射对胃癌病人免疫功能的影响．激光杂志，2001，22，(1)：58～60
27. Agaiby A, Dyson M. The effects of light on T-lymphocytes proliferation in viro. American Society for lasermedicine and surgery abstracts. Laser Surg Med, suppl, 1992(4): 12
28. Shielding TD, Okaner S, Leckey D, et al. The effect of laser irradiation upon human mononuclear leuko-cytes in vitro. American Society for laser medicine and surgery avbstracts. Laser Surg Med, suppl, 1992, 4:11
29. Zheng H, Qin JZ, Xin H, et al. Low incidentlevel doses of He Nelaser activate phagocytic action of mouse macrophages in vitro and in vovo. American Society for laser medicine and surgery abstracts. Laser Surg Med, suppl, 1992 ,(4): 11～12
30. Mc Gowan M, Yu W, Naim JO, et al. The effects of low level laser irradiation on IL-2 release from CD4T- cells. American Society for laser medicine and surgery abstracts. Lasser Surg Med, suppl, 1996, 8:6
31．赵福运．低强度光的生理作用及作用机理．实用激光治疗学，1997，10：253～271
32. Metster E, Siry T, Szende B, et al. Effects of last rats on wound healing. Am J surg, 1971,122:532
33．陈昌钧．低功率氦氖激光和二氧化碳激光治疗山羊局部烧伤性研究．激光杂志，1996，17(2)：90～94
34. Trelles MA, Mayayo E. Bone fracture consolidates faster with low power laser.Lasers Surg Med, 1987, 7(1): 36～45
35. Mavrich VV, Luzin VI. The growth, chemical composition and strength properties of the long tubular bones in the skeleton of white rats under the influence of low-energy laser radiation.Morfologiia, 2000;117(1): 59～66
36. Prikuls VF. Experience in irradiating with helium-neon lasers to treat patients with relapsing aphthous stomatitis.Stomatologiia, 2000;79(6): 20～22
37. Dushin NV, Azibekian AB, Ali Akhsan M. Treatment of lacrimal duct diseases using low energy helium-neon laser. Vestn Oftalmol, 2002,118(2): 15～17
38．罗奇，熊明根，顾浩等．低能量氦-氖激光血管内照射治疗皮肤撕脱伤疗效观察．中国修复重建外科杂志，2000，14(1)：7～9
39．孙万彬，赵春芳，谷双魁等．硫堇块染色法显示神经元尼氏体的体会．四川解剖学杂志，2000，9(2)：86～88
40．韩太真，吴馥梅．学习与记忆的神经生物学．第一版．北京：北京医科大学中国协
和医科大学联合出版社，1998，107～108
41． Tomimoto H, Yanagihara T. Electron microscopic investigation of the cerebral cortex after cerebral ischemia and reperfusion in the gerbil. Brain Res, 1992, 598(1-2) :87-97
42． Deshpande J, Bergstedt K, Linden T, et al. Ultrastructural changes in the hippocampal CA1 region following transient cerebral ischemia: evidence against programmed cell death. Exp Brain Res, 1992, 88(1) :91-105
43． Tomimoto H, Yanagihara T. Golgi electron microscopic study of the cerebral cortex after transient cerebral ischemia and reperfusion in the gerbil. Neurosci, 1994 , 63(4) :957-974
44． Hori N, Carpenter DO. Functional and morphological changes induced by transient in vivo ischemia. Exp Neurol, 1994, 129(2) :279-289
45． 董兰凤，范莉英，苏丽英等．氦氖激光穴位针刺对脾脏作用的电镜观察．针刺研 究，1996，21(4) ：64-67
46． Siposan DG, Lukacs A. Relative variation to received dose of some erythrocytic and leukocytic indices of human blood as a result of low-level laser radiation: an in vitro study. J Clin Laser Med Surg , 2001, 19(2) :89-103
47． Barners CA. L earning and memory: the search for their neurobiological mechanisms in the rat. TINS, 1990, 11(2) : 163-169
48． Tanaka K, Ogawa N, Masato A, et al. Relationship between cholinergic dysfunction and discrimination learning disabilities in Wister rats following chronic cerebral hypoperfusion. Brain Res, 1996, 729 (1) : 55-65
49． 范文辉，刘之荣，李露斯．血管性痴呆的动物模型及其胆碱能机制研究．第三 军医大学学报，2000，22(4) ：314～317
50． Barde YA, Edgar D, Thoenen H. Purification of a new neurotrophic factor from mammalian brain. EMBO J, 1982;1(5) : 549-553
51． Yan Q, Rosenfeld RD, Matheson CR, et al. Expression of brain-derived neurotrophic factor protein in the adult rat central nervous system. Neurosci, 1997, 78(2) :431-448
52． James M, Julie C, Hiao Yan, et al. Distribution of BDNF protein and mRNA in the
normal adult rat CNS. J Neurosci, 1997,17:2295-2313
53． Sendtner M, Holtmann B, Kolbeck R, et al. Brain-derived neurotrophic factor prevents the death of motoneurons in newborn rats after nerve section. Nature, 1992, 24-31;360(6406) : 757-759
54． Kishino A, Ishige Y, Tatsuno T, et al. BDNF prevents and reverses adult rat motor neuron degeneration and induces axonal outgrowth. Exp Neurol, 1997, 144(2) :273-286
55． Kohmura E, Yuguchi T, Yoshimine T, et al. BDNF atelocollagen mini-pellet accelerates facial nerve regeneration. Brain Res, 1999, 849(l-2) :235-238
56． Sherrard RM, Bower AJ. BDNF and NT3 extend the critical period for developmental climbing fibreplasticity. Neuroreport,2001,12(13) : 2871-2874
57． Yamashita K, Wiessner C, Lindholm D, Post-occlusion treatment with BDNF reduces infarct size in a model of permanent occlusion of the middle cerebral artery in rat. Metab Brain Dis, 1997, 12(4) :271-280
58． Tsukahara T, Yonekawa Y, Tanaka K, et al. The role of brain-derived neurotrophic factor in transient forebrain ischemia in the rat brain. Neurosurg, 1994, 34(2) :323-331
59． Larsson E, Nanobashvili A, Kokaia Z, et al. Evidence for neuroprotective effects of endogenous brain-derived neurotrophic factor after global forebrain ischemia in rats. J Cereb Blood Flow Metab, 1999, 19(11) : 1220-1228
60． Endres M, Fan G, Hirt L, et al. Ischemic brain damage in mice after selectively modifying BDNF or NT4 gene expression. J Cereb Blood Flow Metab, 2000, 20(1) :139-144
61． Pringle AK, Sundstrom LE, Wilde GJ, et al. Brain-derived neurotrophic factor, but not neurotrophin-3, prevents ischaemia-induced neuronal cell death in organotypic rat hippocampal slice cultures. Neurosci Lett, 1996, 211(3) :203-206
62． Beck T, Lindholm D, Castren E, et al. Brain-derived neurotrophic factor protects against ischemic cell damage in rat hippocampus. J Cereb Blood Flow Metab, 1994, 14(4) :689-692
63． Kiprianova I, Sandkuhler J, Schwab S, et al. Brain-derived neurotrophic factor
improves long-term potentiation and cognitive functions after transient forebrain ischemia in the rat. Exp Neurol, 1999, 159(2) : 511-519
64． Endres M, Fan G, Hirt L, et al. Ischemic brain damage in mice after selectively modifying BDNF or NT4 gene expression. J Cereb Blood Flow Metab, 2000,20 (1) : 139-144
65． Larsson E, Nanobashvili A, Kokaia Z, et al. Evidence for neuroprotective effects of endogenous brain-derived neurotrophic factor after global forebrain ischemia in rat. J Cereb Blood Flow Metab, 1999, 19(11) : 1220-1222
2. Robert C. Vannucci, Robert M. Brucklacher, et al. Intracellular calcium accumulation during the evolution of hypoxic-ischemic brain damage in the immature rat. Dev Brain Res,2001, 26: 117～120
3. Buonocore G, Perrone S, Muraca MC. Free radicals and brain damage in newborns with hypoxic-ischemic lesion. Ann Ist Super Sanita, 2001,37(4): 527～535
4. Bettina M, Frank P, Pierre G.Effects of interleukin-10 on neonatal excitotoxic brain lesions in mice. Dev Brain Res, 2003,141:25～32
5. Mark R. Pulera, Lisa M, Hantao Liu, et al. Apoptosis in a Neonatal Rat Model of Cerebral Hypoxia-Ischemia.Stroke, 1998,29:2622～2630
6．吴明远，石一复．新生儿缺氧缺血性脑损伤的防治进展．中华妇产科杂志，2000，35(10)：630～632
7. Berger R, Garnier Y.Perinatal brain injury. J Perinat Med, 2000, 28:261～285
8．徐国祥，章萍，唐建民等．激光医学．第一版．北京：人民卫生出版社，1998，6：129～134
9. Mirsky N, Krispel Y, Shoshany Y, et al. Promotion of angiogenesis by low energy laser irradiation. Antioxid Redox Signal, 2002, 4(5): 785～790
10. Reddy GK, Stehno-Bittel L, Enwemeka CS. Laser photostimulation accelerates wound healing in diabetic rats. Wound Repair Regen, 2001, 9(3): 248～255
11. Rochkind S, Nissan M, Alon M, et al. Effects of laser irradiation on the spinal cord for the regeneration of crushed peripheral nerve in rats. Lasers Surg Med,2001,28(3): 216～9
12．于晓虹，华仲慰，李玉书．新生鼠半球性脑缺血的实验模型．第一军医大学
学报，1990，10(3)：236～238
13．华兴邦，李辞蓉．大鼠穴位图谱的研制．实验动物与动物实验，1991，(1)：1～5
14．许能贵，许冠荪，钟平等．电针督脉经穴对急性脑缺血大鼠一氧化氮及内皮素的影响．针刺研究，1996，21(3)：18～20
15．刑艳丽，姚凤祯，杜莹莹．头穴针刺次数对中风病人血液流变学的影响．中国针灸，1994，14(4)；37
16．聂卉．电针对中风偏瘫患者体感诱发电位动态观察．中国针灸，1999，19(6)：369
17．马元．浅谈大椎．针灸临床杂志，1994，10(1)：41～42
18．许能贵，周逸平，许冠荪等．电针大椎、百会穴对局灶性脑缺血大鼠脑血流量和自发脑电的影响．中国中医药科技，2001，8(1)：3～5
19．周筠梅．ATP合成酶的结构变化机理和旋转催化．生物化学与生物物理进展，1998，25(1)：9
20．高震．论经穴能量系统的本质及刺激传导机理．中国针灸，2001，21(3)：161～163
21．王冰水，易南，李玲等．不同功率氦氖激光照射与大鼠脊髓降钙素基因相关肽表达的关系．现代康复，2000，4(6)：858～859
22．王冰水，李玲，易南等．氦氖激光照射对大鼠神经损伤后脊髓内生长相关蛋白表达的影响．中华物理医学与康复杂志，2000，22(2)：89～91
23．罗荣辉，刘婉华，罗先润．氦氖激光内关穴照射对老年冠心病患者血流动力学的影响．河南医科大学学报，2001，36(4)：417～418
24．张卫光，夏家骝，陶平等．氦氖激光照射大鼠心前区对血液流变学指标的影响．中华理疗杂志，2000，23(2)：17～19
25．朱长庚．免疫-神经-内分泌网络．解剖学报，1993，24(2)：216～221
26．张红宇，杨桐伟，王广义等．氦氖激光穴位照射对胃癌病人免疫功能的影响．激光杂志，2001，22，(1)：58～60
27. Agaiby A, Dyson M. The effects of light on T-lymphocytes proliferation in viro. American Society for lasermedicine and surgery abstracts. Laser Surg Med, suppl, 1992(4): 12
28. Shielding TD, Okaner S, Leckey D, et al. The effect of laser irradiation upon human mononuclear leuko-cytes in vitro. American Society for laser medicine and surgery avbstracts. Laser Surg Med, suppl, 1992, 4:11
29. Zheng H, Qin JZ, Xin H, et al. Low incidentlevel doses of He Nelaser activate phagocytic action of mouse macrophages in vitro and in vovo. American Society for laser medicine and surgery abstracts. Laser Surg Med, suppl, 1992 ,(4): 11～12
30. Mc Gowan M, Yu W, Naim JO, et al. The effects of low level laser irradiation on IL-2 release from CD4T- cells. American Society for laser medicine and surgery abstracts. Lasser Surg Med, suppl, 1996, 8:6
31．赵福运．低强度光的生理作用及作用机理．实用激光治疗学，1997，10：253～271
32. Metster E, Siry T, Szende B, et al. Effects of last rats on wound healing. Am J surg, 1971,122:532
33．陈昌钧．低功率氦氖激光和二氧化碳激光治疗山羊局部烧伤性研究．激光杂志，1996，17(2)：90～94
34. Trelles MA, Mayayo E. Bone fracture consolidates faster with low power laser.Lasers Surg Med, 1987, 7(1): 36～45
35. Mavrich VV, Luzin VI. The growth, chemical composition and strength properties of the long tubular bones in the skeleton of white rats under the influence of low-energy laser radiation.Morfologiia, 2000;117(1): 59～66
36. Prikuls VF. Experience in irradiating with helium-neon lasers to treat patients with relapsing aphthous stomatitis.Stomatologiia, 2000;79(6): 20～22
37. Dushin NV, Azibekian AB, Ali Akhsan M. Treatment of lacrimal duct diseases using low energy helium-neon laser. Vestn Oftalmol, 2002,118(2): 15～17
38．罗奇，熊明根，顾浩等．低能量氦-氖激光血管内照射治疗皮肤撕脱伤疗效观察．中国修复重建外科杂志，2000，14(1)：7～9
39．孙万彬，赵春芳，谷双魁等．硫堇块染色法显示神经元尼氏体的体会．四川解剖学杂志，2000，9(2)：86～88
40．韩太真，吴馥梅．学习与记忆的神经生物学．第一版．北京：北京医科大学中国协
和医科大学联合出版社，1998，107～108
41． Tomimoto H, Yanagihara T. Electron microscopic investigation of the cerebral cortex after cerebral ischemia and reperfusion in the gerbil. Brain Res, 1992, 598(1-2) :87-97
42． Deshpande J, Bergstedt K, Linden T, et al. Ultrastructural changes in the hippocampal CA1 region following transient cerebral ischemia: evidence against programmed cell death. Exp Brain Res, 1992, 88(1) :91-105
43． Tomimoto H, Yanagihara T. Golgi electron microscopic study of the cerebral cortex after transient cerebral ischemia and reperfusion in the gerbil. Neurosci, 1994 , 63(4) :957-974
44． Hori N, Carpenter DO. Functional and morphological changes induced by transient in vivo ischemia. Exp Neurol, 1994, 129(2) :279-289
45． 董兰凤，范莉英，苏丽英等．氦氖激光穴位针刺对脾脏作用的电镜观察．针刺研 究，1996，21(4) ：64-67
46． Siposan DG, Lukacs A. Relative variation to received dose of some erythrocytic and leukocytic indices of human blood as a result of low-level laser radiation: an in vitro study. J Clin Laser Med Surg , 2001, 19(2) :89-103
47． Barners CA. L earning and memory: the search for their neurobiological mechanisms in the rat. TINS, 1990, 11(2) : 163-169
48． Tanaka K, Ogawa N, Masato A, et al. Relationship between cholinergic dysfunction and discrimination learning disabilities in Wister rats following chronic cerebral hypoperfusion. Brain Res, 1996, 729 (1) : 55-65
49． 范文辉，刘之荣，李露斯．血管性痴呆的动物模型及其胆碱能机制研究．第三 军医大学学报，2000，22(4) ：314～317
50． Barde YA, Edgar D, Thoenen H. Purification of a new neurotrophic factor from mammalian brain. EMBO J, 1982;1(5) : 549-553
51． Yan Q, Rosenfeld RD, Matheson CR, et al. Expression of brain-derived neurotrophic factor protein in the adult rat central nervous system. Neurosci, 1997, 78(2) :431-448
52． James M, Julie C, Hiao Yan, et al. Distribution of BDNF protein and mRNA in the
normal adult rat CNS. J Neurosci, 1997,17:2295-2313
53． Sendtner M, Holtmann B, Kolbeck R, et al. Brain-derived neurotrophic factor prevents the death of motoneurons in newborn rats after nerve section. Nature, 1992, 24-31;360(6406) : 757-759
54． Kishino A, Ishige Y, Tatsuno T, et al. BDNF prevents and reverses adult rat motor neuron degeneration and induces axonal outgrowth. Exp Neurol, 1997, 144(2) :273-286
55． Kohmura E, Yuguchi T, Yoshimine T, et al. BDNF atelocollagen mini-pellet accelerates facial nerve regeneration. Brain Res, 1999, 849(l-2) :235-238
56． Sherrard RM, Bower AJ. BDNF and NT3 extend the critical period for developmental climbing fibreplasticity. Neuroreport,2001,12(13) : 2871-2874
57． Yamashita K, Wiessner C, Lindholm D, Post-occlusion treatment with BDNF reduces infarct size in a model of permanent occlusion of the middle cerebral artery in rat. Metab Brain Dis, 1997, 12(4) :271-280
58． Tsukahara T, Yonekawa Y, Tanaka K, et al. The role of brain-derived neurotrophic factor in transient forebrain ischemia in the rat brain. Neurosurg, 1994, 34(2) :323-331
59． Larsson E, Nanobashvili A, Kokaia Z, et al. Evidence for neuroprotective effects of endogenous brain-derived neurotrophic factor after global forebrain ischemia in rats. J Cereb Blood Flow Metab, 1999, 19(11) : 1220-1228
60． Endres M, Fan G, Hirt L, et al. Ischemic brain damage in mice after selectively modifying BDNF or NT4 gene expression. J Cereb Blood Flow Metab, 2000, 20(1) :139-144
61． Pringle AK, Sundstrom LE, Wilde GJ, et al. Brain-derived neurotrophic factor, but not neurotrophin-3, prevents ischaemia-induced neuronal cell death in organotypic rat hippocampal slice cultures. Neurosci Lett, 1996, 211(3) :203-206
62． Beck T, Lindholm D, Castren E, et al. Brain-derived neurotrophic factor protects against ischemic cell damage in rat hippocampus. J Cereb Blood Flow Metab, 1994, 14(4) :689-692
63． Kiprianova I, Sandkuhler J, Schwab S, et al. Brain-derived neurotrophic factor
improves long-term potentiation and cognitive functions after transient forebrain ischemia in the rat. Exp Neurol, 1999, 159(2) : 511-519
64． Endres M, Fan G, Hirt L, et al. Ischemic brain damage in mice after selectively modifying BDNF or NT4 gene expression. J Cereb Blood Flow Metab, 2000,20 (1) : 139-144
65． Larsson E, Nanobashvili A, Kokaia Z, et al. Evidence for neuroprotective effects of endogenous brain-derived neurotrophic factor after global forebrain ischemia in rat. J Cereb Blood Flow Metab, 1999, 19(11) : 1220-1222
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