【作者】 富奇志；

【导师】 谢鹏；

【作者基本信息】 重庆医科大学 ， 神经病学， 2010， 博士

【摘要】 研究背景随着电子和生物信息工程技术不断发展,通过BAEP、SEP、MRI等在内的多种方法能够了解脑干做为生命中枢的各项功能及结构变化,但将其作为床旁诊断及动态监测方法尚存在一定的局限性。因此,临床医生迫切需要一种无创、准确、简单、可靠、且能为诊断治疗提供有价值信息的实时床旁监测手段。本研究通过动物实验探讨一种操作简易无创,可客观、准确、特异地对脑干功能损害进行定位的方法,为其将来应用于临床奠定基础。目的1建立不同剂量胶原酶诱导大鼠一侧基底节区脑出血模型,观察其不同时间点颅内压力、自发眼震及冰水诱发眼震电图的变化及脑干组织病理改变三者之间的相互关系,探讨应用眼震电图动态监测脑干功能的可行性。2观察不同剂量胶原酶诱导大鼠一侧基底节区脑出血模型脑干不同层面神经核团及内侧纵束的变化及其与颅内压的关系,探讨一侧基底节区脑出血后自发和冰水诱发眼震异常的病理基础。3比较自发眼震和冰水诱发眼震电图在大鼠不同层面脑干离断损伤模型与大鼠一侧基底节区脑出血模型的异同性,进一步验证脑干损害是一侧基底节区脑出血后自发眼震和冰水诱发眼震异常的病理基础,为眼震电图作为判定脑出血后脑干损伤的手段提供佐证。4观察脑干内眼动相关核团和不同阶段内侧纵束分别被损毁和刺激后自发眼震和冰水诱发眼震电图的变化,进一步阐明一侧基底节区脑出血引起冰水诱发眼震异常和自发眼震的可能病理机制,为眼震电图用于判定脑出血后脑干损伤提供证据。方法第一部分:冰水诱发眼震电图对大鼠一侧基底节区脑出血模型中脑干损害的判定1 Rosenberg法建立脑出血模型:大鼠分为5组,假手术组、生理盐水对照组、0.3UⅦ型胶原酶注入组、0.6UⅦ型胶原酶注入组、0.9UⅦ型胶原酶注入组。2电生理记录:RM6240C多道生理记录仪记录各组大鼠术后12h、24h、48h、72h、5d及7d时颅内压、自发眼震和冰水诱发眼震。3 HE染色观察脑干组织病理改变。4 Pearson法分析颅内压变化、脑干不同层面组织病理学变化及冰水诱发眼震电图的潜伏期(Lat)、频率(F)、波幅(Amp)、慢相角速度(SPV)、节律变异指数(CVI)之间的相关性,判别分析法得到冰水诱发眼震电图各参数对不同层面的脑干损伤的判定函数。第二部分:大鼠一侧基底节区脑出血后自发和诱发眼震异常的机制1一侧基底节区脑出血后脑干神经核团及内侧纵束组织学变化。免疫组化法测定动眼神经核兴奋性和抑制性神经递质受体表达反应脑干损伤分子水平的变化;TUNEL染色测定脑干不同层面的细胞凋亡程度反应脑干损伤细胞水平变化;免疫组化法观察内侧纵束在脑干不同阶段的MBP表达情况反应脑干损伤髓鞘变化。2大鼠不同脑干层面离断模型后自发眼震和冰水诱发眼震电图变化。在不破坏小脑的前提下,采用经小脑延髓脉络膜裂入路充分暴露脑干,分别机械性离断不同的脑干层面:中脑四叠体上;中脑四叠体下;脑桥上段;脑桥中下段。RM6240C多道生理记录仪监测呼吸和血压变化,同时记录自发眼震和冰水诱发眼震电图。术中给予实验大鼠呼吸支持。3大鼠脑干不同核团及脑干内侧纵束不同阶段电凝破坏及电刺激后自发眼震和冰水诱发眼震电图变化。分别电凝损毁和电刺激一侧动眼神经核、脑桥旁正中网状核和前庭神经核;分别电凝损毁脑干不同阶段的内侧纵束:中脑四叠体;中脑四叠体上+中脑四叠体下;中脑四叠体上+中脑四叠体下+脑桥上段;中脑四叠体上+中脑四叠体下+脑桥上段损毁+脑桥中下段。分析比较不同核团受到刺激和破坏后自发眼震及冰水诱发眼震电图各参数的变化;同一核团刺激性病变和破坏性病变眼震电图差异;不同阶段内侧纵束损毁后冰水诱发眼震电图各参数之间的差异。结果第一部分1不同剂量胶原酶注入大鼠一侧基底节区脑出血模型后可以引起不同程度的颅内压水平升高(P<0.01),在造模后48h-72h达到高峰,同期胶原酶注入量越大,颅内压水平越高,组间比较差异显著(P<0.01)。2不同剂量胶原酶注入大鼠一侧基底节区脑出血模型后,在不同时间点可记录到多种类型的自发眼震。所有造模组除0.3U胶原酶注入组12h冰水诱发眼震电图各参数与对照组比较无差异(P>0.05)外,其它各造模组在各时间点均存在差异(P<0.01)。同时间点胶原酶注入量越大,潜伏期越长,频率越慢,波幅越小,慢相角速度越慢,节律变异指数越大(P<0.01)。颅内压越高,冰水诱发眼震潜伏期越长,其波幅越小,频率越慢,慢相角速度越慢,节律变异指数越大(P<0.01)。3 HE染色显示造模组脑干出现组织坏死改变,神经元肿胀,核固缩深染及神经纤维结构紊乱;部分出现炎性细胞聚集和出血。随着注入胶原酶剂量的增加,颅内压的升高,脑干的病理改变呈现由上向下发展,且范围逐步扩大。4判别分析法得到冰水诱发眼震电图中Lat、F、Amp、SPV、CVI五项参数变化对脑干损伤层面定位的正确判断率可达到79.5%。第二部分:1脑干核团及内侧纵束组织学变化:①所有脑出血造模组各时间点双侧动眼神经核的NMDAR1表达与对照组比较均下降(P<0.01),组间水平差异显著(P<0.01),相应GABA-ARα1表达无变化(P>0.05)。同期注入胶原酶量越多, NMDAR1表达越低。②所有脑出血造模组各时间点脑干内凋亡细胞阳性百分率较对照组增多(P<0.01),12h-24h时主要分布在中脑或脑桥上段;48h-72h各阶段脑干细胞凋亡阳性百分率增高(P<0.01),0.6U及0.9U胶原酶注入组脑桥下段凋亡阳性细胞百分率明显增高(P<0.01)。各造模组组间比较同期胶原酶注入量越大,同部位凋亡阳性细胞百分率越高,比较有显著差异(P<0.05)。提示胶原酶注入量越大,脑干细胞的凋亡出现越早,细胞凋亡百分率越高,波及范围越广泛,呈现自中脑向脑桥下段的发展顺序;③造模组大鼠脑干内髓鞘着色变浅,平均光密度下降(P<0.01),且不均匀,束状结构中断,排列紊乱呈网格状,提示存在髓鞘脱失。中脑段内侧纵束平均光密度值在0.3U、0.6U、0.9U胶原酶注入组分别于造模后72h、24h、12h出现下降,在组内随着时间趋势逐步降低,在48h-72h达到最低值,与对照组比较有差异(P<0.01);0.6U及0.9U组分别于造模后72h、48h出现脑桥上段MBP平均光密度值下降,在组内随着时间趋势逐步降低,在48h-72h达到最低值,与对照组比较有差异(P<0.05)。脑桥下段MBP异常改变仅在0.9U造模组后48h出现(P<0.05),呈现自中脑至脑桥下段内侧纵束全段的MBP表达异常。胶原酶注射剂量越大,脑干的MBP表达异常发生越早,平均光密度值越小提示髓鞘脱失越明显,波及范围越广泛,在脑干内呈现自上而下的发展。④颅内压越高,动眼神经核NMDAR1表达水平越低,中脑、脑桥上段和脑桥下段的细胞凋亡比例越高,内侧纵束MBP表达越低,。2不同阶段脑干层面离断后可以出现不同类型的自发眼震,表现形式在中脑四叠上离断为上跳性眼震;中脑四叠体下离断为汇聚性及扭转痉挛性眼震;脑桥上段离断为下跳性眼震;脑桥下段离断为眼球急动和周期性交替眼震。中脑四叠体离断后冰水诱发眼震电图各参数与对照组比较无差异(P>0.05),其它层面离断后冰水实验均未诱发出眼震。3①右侧OMN损毁后双侧冰水诱发眼震电图较对照组比较Lat和F无明显差异(P>0.05),Amp下降,SPV下降,CVI升高(P<0.05)。右侧OMN电刺激后冰水诱发眼震电图参数与对照组比较无差异(P>0.05)。②右侧PPRN破坏后左侧冰水诱发眼震电图与对照组比较形式无差异,其Lat延长,F减慢,Amp下降,SPV减慢,CVI增加(P<0.05),右侧冰水未诱发眼震。右侧PPRN电刺激,右侧冰水诱发眼震与对照组比较眼震的形式无差异,Lat不变, F加快,Amp升高,SPV增加,CVI增加(P<0.05)。左耳灌注冰水诱发眼震与对照组比较眼震的形式无差异,潜伏期不变,但频率减慢,波幅下降,慢相角速度减慢,节律变异指数增加(P<0.05)。③右侧VN破坏后右侧冰水诱发眼震与对照组比较眼震的形式无差异,潜伏期不变,频率增快,波幅加大,慢相角速度加快,节律变异指数增加(P<0.05),左侧冰水未诱发出眼震。右侧VN电刺激后左侧冰水诱发眼震与对照组比较眼震的形式无差异,Lat不变,F增快,Amp加大,SPV加快,CVI增加(P<0.05)。右侧冰水诱发眼震潜伏期不变,F减慢,Amp下降,SPV减慢,CVI增加(P<0.05)④在逐层连续电凝损毁不同阶段的MLF冰水诱发实验中,中脑四叠体上损毁后冰水诱发眼震的Lat、F、Amp、SPV及CVI与对照组比较无显著性差异(P>0.05)。随着下面逐层的破坏,冰水诱发眼震潜伏期延长、频率变慢、波幅下降、慢相角速度减慢及节律变异指数增加(P<0.05)。中脑四叠体上+中脑四叠体下损毁后+脑桥上段损毁+脑桥中下段全部损毁组所有动物冰水试验未诱发眼震。结论大鼠一侧基底节区不同程度脑出血模型在不同时间点出现自发眼震和冰水诱发眼震不同的改变,是因为不同程度颅内压升高引起脑干眼动相关核团及内侧纵束功能损害所致。冰水诱发眼震电图相关参数的特征性改变及自发眼震可作为判定脑干损伤部位及程度的重要依据。更多还原

【Abstract】 BackgroundCurrently with the developing of the biological information technology, the means of monitoring brainstem function are improved and are diversified gradually, including BAEP, SEP and MRI and so on. But as bedside diagnostic monitoring tools, they still had some limitations. Therefore, the study will investigate a clinical bedside monitoring method which can be operated non-invasively and simply. It can be used to help the clinician to judge the brainstem function lesion objectively and accurately. The study will construct a foundation for its further clinical application.Purposes1 Through constructing the experimental basal ganglia intracerebral hemorrhage model in rats induced by different doses of collagnase, to observe the changes of intracranial pressure, pathological changes of different brainstem segments, spontaneous nystagmus and ice water-induced ENG and the relationships among them. It was achieved that the feasibility of the ENG being applied to monitor the function of brainstem dynamicly.2 Through observing the changes of nucleus and the medial longitudinal fasciculi in brainstem and the relationships with ICP, the pathological bases were explored how the spontaneous nystagmus and the abnormal nystagmus induced by ice water test occurred after the experimental basal ganglia intracerebral hemorrhage model in rats induced by different doses of collagnase.3 To further verificate brainstem damage being the cause of spontaneous nystagmus and the abnormal ENG ice water-induced after the experimental basal ganglia intracerebral hemorrhage, the similarities and differences of spontaneous nystagmus and ice water-induced ENG were compared between the rat models of which the brainstems were transected at different levels and of the basal ganglia intracerebral hemorrhage. It could provide evidences for ENG being applied to judge the brainstem damage after ICH.4 To explore the mechanisms of spontaneous nystagmus and the abnormal ENG ice water-induced caused by brainstem damage, the changes of spontaneous nystagmus and ENG ice water-induced were observed after the brainstem nucleis and the medial longitudinal fasciculus in different stages being destructed and stimulated respectively. It could provide evidences for ENG being applied to judge the brainstem damage after ICH. MethodsPart I: The value which ice water-induced ENG was used being the method to judge the brainstem damage after the experimental basal ganglia intracerebral hemorrhage in rats.1 The experimental basal ganglia intracerebral hemorrhage (ICH) was set up according with the Rosenberg’s way. Rats were divided into five groups: sham group, saline control group, 0.3U collagenase injection group, 0.6U collagenase injection group, 0.9U collagenase injection group.2 Electrophysiological recording: RM6240C multi-channel physiol- ogical recorder recorded the curve of the intracranial pressure, the spontaneous nystagmus and the ENG ice water-induced at 12h, 24h, 48h, 72h, 5d and 7d after ICH.3 Pathological changes of brainstem was observed through HE staining.4 Intracranial pressure, pathological changes of the different brainstem segments and ENG ice water-induced including latency (Lat), frequency (F), amplitude (Amp), slow-phase velocity (SPV), circadian variation index (CVI) were given correlation analysis. The function was detectived that the parameters of ENG ice water-induced was applied to judge the different segments of brainstem damage through discriminant analysis.Part II: The occurring mechanism of the spontaneous nystagmus and the abnormal ice water-induced ENG after the experimental basal ganglia intracerebral hemorrhage in rats.1 The expression levels of NMDAR1 and GABA-ARα1 in oculomotor nucleus (OMN) were detected by immunohistochemistry being responsed to the changes of neuron in the molecular level. TUNEL staining determined the degrees of apoptosis at different brainstem segments being responsed to the changes of neuron in cellular level. Immunohistochemical method measured the MBP expression level of medial longitudinal fasciculus in the brainstem at different layers being responsed to the changes of brainstem myelin.2 The changes of spontaneous nystagmus and ENG ice water-induced after the different layers of brainstem were transeced in rats. The brainstem was exposed fully through the enter way of the cerebellomedullary choroidal fissure which the cerebellum was intact. The brainstem was transected at different layers: superior to midbrain quadrigeminal body, inferior to midbrain quadrigeminal body, superior pons, inferior pons. Changes of respiration, blood pressure, spontaneous nystagmus and ENG ice water-induced were also recorded by RM6240C multi-channel physiological recorder.3 The changes of spontaneous nystagmus and ENG ice water-induced after the different nucleis and the different stages of the medial longitudinal fasciculus were electric coagulated damaged and electrical stimulated. One lateral oculomotor nucleus, paramedian pontine reticular nucleus and vestibular nucleus were damaged by electrocoagulation and electric stimulated respectively. The different stages of medial longitudinal fasciculus were electrocoagulated at the layer of superior midbrain quadrigeminal body, the layers of superior and inferior midbrain quadrigeminal body, the layers of superior and inferior midbrain quadrigeminal body and superior pons, the layers of superior and inferior midbrain quadrigeminal body, superior pons and inferior pons. Spontaneous nystagmus and parameters of ENG ice water-induced were analyzed and compared among the different nucleis being damaged or stimulated, so as to the different layers of the medial longitudinal fasciculus being damaged.ResultsPart I:1 The level of ICP was normal in 0.3U collagenase injected group at 12h, rose at 24h and reached the climax at 48h-72h. ICP of 0.6U and 0.9U collagenase injected group were heightened at 12h and reached the peak at 48h-72h (25mmHg). The more the collagenase were injected the ICP were higher at same time.2 Different style spontaneous nustagmus were recorded at different period after ICH. There had significant difference between ICH groups and control group in the parameters of ENG ice water-induced at each time (P<0.01), except for 0.3U collagenase injected group at 12h (P>0.05). The more the dosage of collagenase was injected, the sooner the changes of ice water induced nystagmus occured, the longer the latency was, the slower the frequency was, the smaller the amplitude was, the slower the slow-phase velocity was, the greater circadian variation index was (P<0.01).3 Pathological changes of brainstem: HE staining showed that the neurons being swelling , the nucleus being condensed and deep-dyed and the neuronal fibers structurel being disordered. The accumulation of inflammatory cells and bleeding also could be seen. The higher dose of collagenase being injected, the higher ICP elevated, the more obviously the pathological changes occurred in brainstem. The developing sequence was from up to down within brainstem.4 The accurate rate to judge the brainstem lesion localization with parameters of ENG ice water-induced including Lat, F, Amp, SPV, CVI could reach 79.5% through discriminant analysis.Part II:1①The NMDAR1 expression levels in OMN of ICH model groups were less than that of control group (P<0.01) at each time. The more the dosage of the collagenase injected, the lower its expression level was and the expression level of GABA-ARα1 had no difference (P>0.05) compared with that of control group.②The percentage of positive apoptotic cells within the brainstem in all ICH groups were higher than that of the control groups (P<0.01). The apoptotic cells were located in the upper layers of the midbrain or pons at 12h-24h after ICH. Then percentage of positive apoptotic cells at all layers of brainstem increased at 48h-72h (P<0.01). The percentage of positive apoptotic cells at inferior pons were increased in 0.6U and 0.9U collagenase injection groups (P<0.01). Comparing among the ICH groups at the same period, the greater the dose of collagenase being injected, the higher the percentage of apoptotic cells was (P<0.05). The results suggested that the greater the doses of collagenase being injected, the earlier the cell apoptosis of brainstem occured, the higher the percentage of apoptotic cells was and the larger the range of apoptosis was involved. The developing sequence was from the midbrain to inferior pons.③It could be seen that coloring of myelin being shallow which bundle structure was disrupted and the average optical density decreased in all ICH groups (P<0.01). It indicated the presence of demyelination. The average optical density of MBP of medial longitudinal fasciculus in the brainstem declined at 72h, 24h, 12h after 0.3U, 0.6U, 0.9U collagenase being injected respectively. The trend showed degrading over time and dropped to the lowest point at 48h-72h compared with the control group (P<0.01). The average optical density descending of MBP in upper pons appeared in 0.6U and 0.9U group at 72h and 48h respectively. There had significant difference within groups (P<0.01). Abnormal expression of MBP in inferior pons only was showed in 0.9U group at 48h (P<0.05), which showed the abnormal expression of MBP of the whole brainstem MLF from the midbrain to pontine. The higher dose of collagenase being injected, the smaller the average optical density of the MBP was, the more obviously the demyelination occurred. The developing sequence was from up to down within brainstem also. Correlation analysis showed that the higher the intracranial pressure was, the lower level of NMDAR1 in the OMN expressed, the lower level of MBP of MLF expressed and the higher level of cell apoptosis within brainstem was.2 Different types of spontaneous nystagmus appeared after the different brainstem segments being transected. The types of spontaneous nystagmus were upbeat nystagmus or torsion spasm nystagmus after the superior midbrain quadrigeminal body being transected. The types of spontaneous nystagmus were downbeat nystagmus after the superior pons being transected. The types of spontaneous nystagmus were eye snap or periodic alternating nystagmus after the inferior pons being transected. All parameters of ENG ice water-induced after midbrain transection had no significant difference compared with the control group (P>0.05). Nystagmus couldn’t be induced by ice water after other brainstem segments being transected.3①There was no difference at Lat and F of ENG ice water-induced between the group which the right lateral OMN being damaged and control group (P>0.05), while the Amp and the SPV descended, the CVI increased (P<0.05). The parameters of ENG ice water-induced while the right OMN being stimulated had no difference compared with the control group (P>0.05).②There was no difference in the type of ENG left lateral ice water-induced after the right PPRN being damaged compared with the control group, but the Lat extended, the F and Amp were down, the SPV was slow, the CVI increased (P<0.05). Nystagmus couldn’t be induced by right ice water test. There was no difference in the type of ENG right lateral ice water-induced after the right PPRN being stimulated compared with the control group, same as the Lat, while the F, the Amp, the SPV and the CVI increased (P<0.05). While the left water induced ENG showed the F, the Amp, the SPV decresed and the CVI was greater(P<0.05).③There was no difference in the type of ENG right lateral ice water-induced after the right VN being damaged compared with the control group, same as the Lat, the Amp, the SPV and the CVI increased (P<0.05). Nystagmus couldn’t be induced by left lateral ice water test. There was no difference in the type of ENG left lateral ice water-induced after the right VN being stimulated compared with the control group , same as the Lat, while the F, the Amp, the SPV and the CVI increased (P<0.05).④After the MLF of superior midbrain quadrigeminal body being electric coagulated, the Lat, F, Amp, SPV and the CVI of the ENG ice water-induced were same as those of the control group (P>0.05). Along with the more layers being destructed from superior to inferior, the latency was extened, the frequency and slow phase velocity became slower, the amplitude descended and the CVI increased about the ENG ice water-induced (P<0.05). The nystagmus induced by ice water test disappeared after the whole MLF of brainstem being damaged.ConclusionsSpontaneous nystagmus and nystagmus ice water-induced could occur different changes at different periods after the basal ganglia ICH model in rats. The reasons were the damages of the nucleus and MLF about the eye movements in brainstem caused by the different degree increased ICP. On the contrary, the locations and the degrees of brainstem damage could be judged according with parameters of ENG ice water-induced and spontaneous nystagmus更多还原