【作者】 潘燕；

【导师】 夏作理； 陈连璧；

【作者基本信息】 山东大学 ， 生理学， 2011， 博士

【摘要】 脑脊液(cerebrospinal fluid, CSF)维持着中枢神经系统(central nevous system,CNS)电解质环境与酸碱平衡,作为一种介质为神经元和神经胶质细胞提供营养,并承担着细胞代谢产物及神经递质、释放因子、神经肽、激素及血浆蛋白等大分子物质的运输与清除工作。大脑的淋巴引流对维持CSF的正常循环及脑内环境的平衡具有重要作用。许多研究已证明,脑内淋巴液经由神经周围淋巴管和血管周围的淋巴管前淋巴系统再经颈部淋巴引流到颅外。引流途径的任一环节受阻均可引发CSF循环不畅及脑内物质的引流受阻,从而造成神经系统内环境的紊乱及神经功能异常,最终导致淋巴性脑水肿(lymphatic brain edema, LBE)及/或淋巴性脑病(lymphatic encephalopathy, LE)的发生。肿瘤治疗过程中的颈部淋巴结清除手术及放射治疗等是引发人类LE的主要原因。人类LE表现为淡漠,自主活动减少,记忆力下降,精力不能集中及头痛、眩晕、恶心与呕吐等症状,严重时可发生惊厥。随着老龄人口的不断增加与环境污染的日益严重,近年来肿瘤的发生率不断增长,接受肿瘤治疗导致的颈淋巴系统功能障碍的人数也在不断增加。由于对LE的认识尚不十分清楚,常导致LE的误诊,从而影响到疾病的治疗。LE引起的脑缺血缺氧损伤还可影响到其对外周组织与器官的调节功能,常常会加重患者原有疾病的病情与临床表现,影响医生对患者病情的判断。近年来已有学者发现LE大鼠出现自主神经系统(autonomic nervous system, ANS)调节能力的改变,引起血压与心率等调节能力下降等,但对其研究尚不够系统与深入。对LE研究的不足,也使得对其治疗缺乏有效的方法与药物。因此充分了解LE的发生、发展过程及其对脑组织和外周器官的病理危害以及寻找有效的缓解或治疗LE的药物与方法将具有重要意义。羟基红花黄素A (hydroxysafflor yellow A, HSYA)是由红花中提取的黄色无结晶粉末。对缺血缺氧造成的神经功能损伤有明显的保护作用,已广泛应用于心脑血管缺血的治疗。其机制与减少脂质过氧化物生成,提高超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性,提高内皮型一氧化氮合酶(eNOS)蛋白表达,减少细胞凋亡,减轻神经组织结构损伤等作用相关。但由颈淋巴梗阻导致的LE属于全脑的损害,HSYA是否对LE造成的脑损伤具有一定的保护作用尚末见研究报道。心率变异性(heart rate variability, HRV)是指连续心搏间瞬时心率的微小涨落。HRV信号蕴含了有关心血管调节的大量信息,广泛地应用于评价ANS系统对心血管功能的调节能力及对心梗后死亡的发生进行预测。对这些信息的提取和分析可以定量评估心脏交感神经和迷走神经活动的紧张性、均衡性及其对心血管系统活动的影响。HRV分析常用的指标包含时域与频域两部分。时域分析通过统计学离散趋势分析法分析R-R间期的变化。频域指标反映交感神经与迷走神经的活动水平及均衡性的变化。其中低频段能量(LF)反映交感神经调制的强度、高频段能量(HF)反映迷走神经调制强度、能量比LF/HF反映交感神经/迷走神经调节平衡。尽管已有学者对LE造成的心血管调节能力损伤进行了一定的研究,但通过标准的心率变异分析工具,全面的评价LE造成的HRV改变尚未见报道。冠心病、心肌梗塞是老年人的常见病与多发病,年老本身亦可引起淋巴引流的障碍,加上老年人口肿瘤发生的比例较高,因肿瘤治疗造成的颈淋巴损伤的比例也会相应地会增大。因此弄清LE对HRV的影响,对准确的判断心梗合并LE患者的病情及预后意义重大。已有报道发现延髓头端腹外侧区(rostral ventrolateraln medulls, RVLM)通过神经递质或神经调质参与心率变异的调节过程。RVLM区域与呼吸节律相关的神经元的放电与HRV的高频成份有关。但目前为止尚未见对LE导致的RVLM区结构的变化进行观察的报道；对其形态学改变与HRV改变间关系的研究亦未见报道。本文试图从ANS调节能力、形态学改变及分子生物学变化等角度探讨LE造成的脑损伤及HSYA对LE的保护作用。希望能够进一步了解LE对ANS调节的影响及寻找改善或治疗LE症状的药物。研究目的1.综合评价LE对ANS调节功能的影响；2.观察LE对RVLM形态结构的影响,了解该区域的损伤情况与损伤程度；3.探讨RVLM结构损伤与HRV改变的关系；4.观察HSYA处理对LE的保护作用；5.探讨HSYA的ANS保护作用机制。研究方法1.实验动物及分组成年雄性Wistar大鼠,体重250-300g,180只。随机分成假手术组(SHAM)、淋巴性脑病(LE)组及羟基红花黄素A (HSYA)组,每组60只。SHAM组只分离暴露淋巴结不实施结扎与摘除手术,不接受任何药物;LE+HSYA组于LE术后每天腹腔注射5mg/kg HSYA;LE组与LE+HSYA组接受同样手术,但术后每天腹腔注射等量生理盐水。上述三组又分为术后1天、3天、5天、7天、14天、21天六个时间点,每个时间组10只。2.LE动物模型的建立应用改良的Casley-smith方法建立LE模型。具体操作如下：戊巴比妥钠腹腔麻醉(30mg/kg),沿颈部正中切开皮肤,分离两侧颈浅淋巴结(各3-5个),结扎其两端的输入、输出淋巴管,摘除淋巴结。然后逐层分离皮下组织、肌层,直至气管。在甲状软骨旁、颈总动脉与迷走神经的后外侧分离出颈深淋巴结(各1-2个),结扎其两端的淋巴管,摘除颈深淋巴结,逐层缝合切口。3.行为学分析实验动物在手术结束后的1、3、5、7、14、21天分别进行行为学评分。评分方法参照全脑缺血模型Lemay法分级标准及Sun等的分级方法并加以调整。4.心电信号提取与HRV分析各组分别于术后1天、3天、5天、7天、14天、21天用戊巴比妥钠腹腔麻醉(30mg/kg)。心电传感器连接肢体Ⅱ导联记录心电信号,温度传感器置入肛门监控体温。使用AD Instrument PowerLab system,采样率2kHz,持续采样30分钟。取其中平稳的5分钟信号进行短时心率变异分析。调用HRV分析模块,对标准肢体Ⅱ导联心电信号进行HRV分析。提取具有代表性的参数包括：心率变异时域指标MEAN (全程所有R-R间期的平均值)、SDNN(全程所有R-R间期的标准差)、RMSSD (全程相邻R-R间期差值的均方根)及频域指标LF(低频能量,0.04<LF<0.15Hz)、HF(高频能量,0.15<HF<0.4Hz)与LF/HF(低高频能量比)。5.组织学观察上述各组每组3只于心电信号测量完成后取RVLM作石蜡切片,行H&E染色光镜观察。并对该区域行超薄切片作电镜观察。6. Real time-PCR及Western Blot应用Real-time PCR及Western blotting技术检测LE对各组大鼠脑组织RVLM内皮型一氧化氮合酶(eNOS)表达的影响及HSYA对eNOS表达的作用。7.数据统计分析：数据的统计分析采用SAS V9.0Chinese。利用UNIVARIATE过程对HRV各指标进行了正态性检验。神经功能缺损得分与心率变异指标的差异性检验,采用基于Wilcox得分的一维非参数检验及对各参数的秩次进行的二维比较与Q检验。结果1.对大鼠行为学指标的影响SHAM组未发现神经功能缺损的表现。与SHAM组比较,LE组的神经功能缺损得分显著偏高(p<0.05),主要表现为大鼠毛发蓬乱缺少光泽、嗜睡、活动减少、肌肉张力降低等；这些现象在术后第三天即可明显被察觉,但通常到第21天消失。LE+HSYA组的神经功能缺损得分比LE组明显降低(p<0.05)。2.对HRV指标及ANS调节功能的影响时域指标：MEAN:与SHAM组比较,LE组自术后第3天起显著延长；LE+HSYA组则更接近于SHAM组水平,且与LE组比较术后3、5、7、14及21天均有显著差异。SDNN与SHAM组比较,LE组术后第3至14天显著增加,而LE+HSYA组增加不明显,与LE组间有显著差异。RMSSD:与SHAM组比较,LE组术后第1至14天均显著增加,而HSYA治疗能显著阻止由LE造成的RMSSD的增加。频域指标：LF：在各时间点三组间均未表现出显著性差异。HF:LE组自术后第3天即比SHAM组显著增大,直至术后第14天,第21天达SHAM组水平；HSYA处理组在术后第5天至14天可抵消由LE造成的HF的增加。LF/HF:LE组较SHAM组术后3至14天显著降低,LE+HSYA组与LE组比较有显著差异,HSYA能显著改善由LE导致的比值的降低。3.对脑组织形态及结构的影响HE染色与光镜观察：SHAM组神经细胞分布均匀、边界清晰,胞核大而规则,呈圆形或椭圆形,色淡,核仁清晰；血管正常。而LE组大鼠神经细胞分布稀疏,体积缩小,排列不整齐。部分神经元、神经胶质细胞肿胀、变性及坏死；有些神经元周围、胞质内有空泡；血管外膜与周围脑组织出现明显空隙,血管外膜水肿。上述变化于LE术后自第3天开始出现,第5-7天最为明显,以后逐渐改善。HSYA治疗组与相应LE组比较HE染色结果均有不同程度的改善。超微结构观察：SHAM组未发现神经细胞凋亡,髓鞘结构致密、排列整齐,血管内皮细胞正常,基底膜完整。LE组与SHAM组形成鲜明对比,表现为核固缩、染色质浓染、核膜不清晰；髓鞘疏松与破裂；毛细血管基底膜肥厚、小血管壁外膜呈半月形或不规则形扩张,内含大量水肿液。上述改变术后第7天最明显。HSYA可显著减少凋亡的神经元的数量和髓鞘及血管的损伤。4.对eNOS蛋白表达的影响与SHAM组比较,LE组eNOS mRNA表达于术后第5、7天显著降低(P<0.05),而HSYA治疗能显著增加eNOS mRNA的表达(P<0.05)。Western blotting结果显示,LE组eNOS蛋白表达显著下调,而HSYA治疗能显著对抗由LE引起的eNOS蛋白表达的下调。结论1.LE可引起ANS调节能力的损伤,使交感迷走平衡被打破,迷走神经在交感迷走平衡中占主导地位,表现为心率降低、HRV增加。2.LE可导致RVLM区域神经元、毛细血管及髓鞘等结构的损伤。3.LE大鼠RVLM区域损伤发生的时间与程度与HRV改变的发生时间与程度密切相关,说明LE所致的ANS调节能力的损伤与RVLM区结构的损伤密切相关。4. HSYA能显著改善LE大鼠的ANS调节能力、改善RVLM区结构的损伤程度、减弱LE下调eNOS mRNA与蛋白表达的影响。表明HSYA (?)(?)够有效的保护LE导致的脑损伤,其作用机制可能部分与NO途径有关。更多还原

【Abstract】 The Cerebrospinal fluid (CSF) maintains electrolyte and acid-base balance in central nervous system (CNS) and furnishes the neurons and glial cells. It also transports and removes the cell metabolites, neurotransmitters, releasing factors, neuropeptide, hormone and cerebral plasma proteins. The lymphatic drainage in the brain plays an important role in keeping the circulation of CSF and the balance of the internal environment of the brain. Previous studies have proved that brain lymph is drained by per neural lymphatic and perivascular lymphatic, after which be drained extra-cranially via cervical lymph. Blocking any part of this pathway can result in turbulence of cerebrospinal fluid circulation and ventricular drainage, causing disturbance of internal environment of the nervous system and abnormal nerve function. As a result, it may cause Lymphatic Brain Edema (LBE) and Lymphatic Encephalopathy (LE). The excision of cervical lymph nodes and radiation therapy during cancer treatment is the primary cause of the Lymphatic Encephalopathy. Human LE shows symptoms such as reduction of locomotor activity, apathia, hypomnesia, headache, dizziness, nausea, vomiting, and convulsion.With the increase in aging population and the severity of pollution, the incidence of cancer has been increasing, so is the number of patients having cervical lymphatic diseases resulted from cancer treatment. Due to limited knowledge on LE, misdiagnoses are not uncommon. In addition, ischemia and hypoxia of cerebral structure due to LE would impact its regulatory function to tissue and organs. In recent years, scholars have found that LE rats present alteration of autonomic nervous function, however, the mechanism of which is still unknown. The lack of knowledge of LE also leads to failing in its treatment. Consequently, fully understanding of the occurrence, development and pathology of LE, and discovering effective therapeutic method for LE will possess great significance.Hydroxysafflor yellow A is yellow amorphous powder which is extracted from the safflower. It has a significant protective effect against the neurological impairment caused by hypoxia and ischemia, and it is widely used for treatment of cerebrovascular and cardiovascular diseases. The underlying mechanisms involve the reduction in the lipid peroxidation, the suppression of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, the up-regulation of the expression of endothelial nitric oxide synthase (eNOS) protein, and the decrease of cell apoptosis and the structural damages at the nervous tissues. However, we are not aware whether HSYA could protect against LE-induced brain injury.Heart rate variability (HRV) is the minimum fluctuation of instantaneous heart period and it is widely used to indirectly assess the regulatory roles of the autonomic nervous system (ANS) in the cardiovascular function. Extracting and analyzing HRV signal can evaluate the contraction, proportion and influence to cardiovascular system, which are created by cardiac sympathetic nerve and cardiac vagus nerve. The common analytical indicators of HRV contain two parts, time-domain and frequency-domain respectively. Time-domain means analyzing the variation among R-R interval via tendency of dispersion approach. Frequency-domain indicator reflects contraction and proportion created by cardiac sympathetic nerve and cardiac vagus nerve, in which low frequency (LF) indicates cardiac sympathetic nerve intensity, high frequency (HF) indicates cardiac vagus nerve intensity, LF/HF indicates relative value between cardiac sympathetic nerve and cardiac vagus nerve. Although scholars have done some research on the cardiovascular regulation injury caused by LE, evaluation of the variability of heart rate thoroughly through a normal analysis tool has not yet been reported. Coronary heart disease, myocardial infarction is a common and frequently occurring disease of the elderly. In addition, aging itself can cause turbulence of lymphatic drainage and incidence of tumor is high among the older people.It has reported that rostral ventrolateral medulls (RVLM) take part in heart rate adjustment through the medium of neurotransmitter or neuromodulator. RVLM is relevant to neural discharge dominated respiratory rhythm and high frequency elements of HRV. But nobody has yet observed the structure change in the RVLM caused by LE and the research which related to its morphology change and heart rate variability has not been reported.The purpose of this study was to investigate LE-induced brain changes in rats by using a combination of behavioral assessments, histological, ECG recordings, real-time PCR and western blot techniques, and to study the protective effects of HSYA.Objective1. Comprehensive understand and evaluate the effects of the regulatory function of autonomic nervous system caused by LE.2. To observe the effects of shape structure in the rostral ventrolateral medullas, and to understand the damage situation and the extent of damage in this region;3. To investigate the relations between structure damage in the rostral ventrolateral medullas and changes of the heart rate variability;4. To investigate the protective effects of HSYA treatment against LE-induced brain injury;5. To discuss the protective function mechanism of hydroxyl safflower yellow A on the autonomic nervous system. Materials and Methods1. Animals and setting of groupsAdult male Wistar rats, weighing250-300g, were obtained from Experimental Animal Center, Shandong University, China. The total180rats were used in this study and randomly divided into three groups. Animals in SHAM-operated group (SHAM; n=60) were subjected to lymphatic nodes exposure surgery without the removal of lymphatic nodes and cervical lymphatic occlusion or any pharmacological treatment. The60rats in HSYA group were given5mg/kg HSYA via intraperitoneal injection once per day after operation. The other60rats in lymphostatic encephalopathy group (LE) received the same surgical procedures as animals in HSYA group but administered an equivalent dose of saline instead of HSYA. Each group was further divided into six subgroups according to the days1,3,5,7,14and21after operation. There were10animals in each subgroup.2. Induction of CLB rat modelsThe CLB model was induced by a modified method of Casley-smith. Briefly, the rats were anesthetized with pentobarbital sodium (30mg/kg) intraperitoneally. An incision of the skin in the neck was made along the midline, The bilateral shallow lymphatic nodes(3-5) were exposed and removed after ligating their afferent and efferent tubes,The trachea was exposed after splitting of subcutaneous tissue and musculature,until the trachea. The deep cervical lymphatic nodes(1-2) could be found at the lateral to thyroid cartilage and posterolateral to the common carotid artery and the nervous vagus. The afferent and efferent lymphatic tubes were ligated, and the lymphatic nodes were then removed.3. Behavior analysisOn postoperative days1,3,5,7,14, and21, the behavior score of each experimental animals was evaluated respectively. Scoring method according to the modified cerebral ischemia model Lemay Lemay and Sun Y grading standards. 4. ECG recordings and analysis of HRV signalsWith0.3%sodium pentobarbital intraperitoneal anesthesia(1ml/100g) on postoperative days1,3,5,7,14and21respectively. The electrocardiogram (ECG)(limb lead Ⅱ) was recorded with ECG sensor, the body temperature was monitored with temperature sensor. The signals were digitized at a sampling rate of2kHz and continuously recorded for30min using a AD Instrument PowerLab system. Then a duration of5-min stable signal was selected to be short-term HRV analyzed. The standard limb lead Ⅱ ECG was selected to be HRV analyzed using HRV analysis module. Extracting representative parameters include:the time-domain indices MEAN of HRV (the mean of all the RR interval throughout)、SDNN (the standard deviation of IBI of all data)、RMSSD (the square root of the mean of the sum of the squares of differences between successive IBI) and the frequency-domain indices:LF (the low frequency power,0.04<LF<0.15Hz); HF (the high frequency power,0.15<HF<0.4Hz) and LF/HF (ratio).5. Histological studyFor hematoxylin and eosin (H&E) staining, the RVLM were separated from three rats of each group after ECG measurement. And the RVLM was used for electron microscopy.6. RT-PCR and Western BlotRT-PCR and western blotting were used to test the eNOS expression in RVLM of LE rats, and the effects of HSYA on the eNOS expression.7. Statistical analysisSAS V9.0Chinese was used for statistical analysis. The positive normal distribution of all HRV parameters was tested by using UNIVARIATE procedure. The differences between LE and SHAM group, as well as LE and HSYA group at each time point were compared using one-way non-parameter test based on Wilcoxon score. Results1. Effects on rats behavioralAnimals in SHAM group displayed no behavior missing. The score of neurological deficit in LE group were significantly higher than that of the SHAM group (p<0.05), rats in LE group exhibited less grooming, drowsiness and less motility than those in SHAM group, which occurred on day3after the CLB operation, and returned to the normal on postoperative day21. However, animals treated with HSYA had lower neurological deficit scores, as compared with those in LE group(p<0.05).2. Effects on heart rate variability indicator and regulation of autonomic nervous system function.Time-domain indicator:MEAN:compared with SHAM section, LE section protracted remarkably since the third day after the surgical operation. There is no significant difference between HSYA and SHAM section. However, compared with LE section, there is significant difference after surgical operation3,5,7,14and21day respectively. HSYA showed recovery in various levels.SDNN:compared with SHAM, LE section protracted remarkably from the third day to the fourteen day after operation, However, HSYA protracted little, so there is significant difference between HSYA and LE section.RMSSD:compared with SHAM, LE section protracted remarkably from day1to day14after operation, and HSYA treatment can block the RMSSD increase owing to LE remarkably.Frequency-domain indicator:LF:There is no significant difference among HSYA SHAM and LE during time points.HF:LE section had been protracting remarkably from day3to day14, and achieves SHAM level at day21. HSYA section can balance out the HF increase created by LE during postoperation days, definitely from day5to day14.LF/HF:compared with SHAM, LE section descended remarkably from day3to day14. There is significant difference between HSYA and LE section, HSYA can ameliorate the diminution as a result of LE.3. Effects on morphology and structure of brain tissueHE staining and light microscopy:In SHAM group, neurons and glial cells were distributed evenly with a clear boundary, the nucleus of neuron were large, round and lightly stained, nucleolus were clear and deeply stained. In contrast, neurons in LE group were sparsely distributed and irregularly arranged. Some neurons and glial cells were swollen and degenerated. Some had vacuoles around the neurons or in the cytoplasm. And the tunica adventitia of blood vessels was swollen and had interspace with cerebral tissue. These changes occurred on day3after CLB operation, and became obviously at postoperative day5and7, and then recovered gradually. HSYA treatment reduced LE-induced histopathological abnormalities.Ultrastructure observe:No apoptotic neurons were observed in SHAM group. In contrast, neurons in LE group showed that highly condensed nuclei, deeply stained chromatin, blurred nuclear membrane. In addition, the myelin sheath in LE group became loose or disintegrated. HSYA administration reduced the number of apoptotic neurons and reduced injury.4. Effects on eNOS expressionThe mRNA expression of eNOS was significantly decreased in LE group, as compared to that in SHAM group at postoperative day5and7(P<0.05), respectively. Treatment with HSYA significantly increased the mRNA expression of eNOS (P<0.05). This result was further confirmed by western blotting at protein levels. The eNOS protein expression in LE group was significantly down-regulated whereas up-regulated at each time point in HSYA-treated group. Conclusion1. LE can damage regulatory function of autonomic nervous system, reduce heart rate, increase heart rate variability and disturb the balance of Sympathovaga, causing vagus nerve dominance.2. LE can cause neuron, capillary and myelin sheath damaged in the RVLM.3. The structural damages of the RVLM appeared to be closely related to the changes of HRV at each time point. Thus, in LE rats, the dysfunction in the regulation of ANS might be due to the impaired structure in the RVLM.4. Treatment with HSYA can remarkably improve the regulatory function of ANS and the impaired structure in the RVLM and it can increase the LE-induced decreasing in eNOS mRNA and protein expression. Our study demonstrated that treatment with HSYA can effectively reduce the brain injury caused by LE and the mechanism might be partly attributed to the nitric oxide pathway.更多还原