【作者】 杨运俊；

【导师】 程敬亮；

【作者基本信息】 郑州大学 ， 影像医学与核医学， 2011， 博士

【摘要】 第一部分大鼠MCAO后远隔部位表观弥散系数的改变背景和目的：缺血性脑血管病(ischemic cerebrovascular disease, ICVD)严重危害着人类的健康及生存质量。根据最新的流行病学调查结果显示,我国ICVD发病率为116/10万,死亡率为81/10万,患病率为3‰,并且呈明显的上升趋势,因此早期诊断和早期治疗至关重要。ICVD后可引起远隔区域(如丘脑及小脑)的功能抑制,其发生机制目前还不甚清楚,可能与神经机能联系不能学说有关。近年来,随着磁共振成像(Magnetic resonance imaging, MRI)新技术的发展,尤其是磁共振弥散加权成像(Diffusion weighted imaging, DWI)技术的出现,使得MRI技术在诊断ICVD方面向着更敏感、更特异方向迈进；但以往人们多偏重于表观弥散系数(Apparent diffusion coefficient, ADC)值对ICVD病灶本身及病灶周边缺血半暗带(Ischemic penumbra, IP)的应用价值,而对于幕上缺血性脑梗死后远隔部位继发性变化的研究,国内外则尚未见相关报道。为了更好地对人类脑梗死疾病进行研究,建立重复性高、稳定性强且脑血管特征与人类及其相似的大鼠脑缺血模型就显得极为重要。应用线栓法制备大脑中动脉阻塞(Middle cerebral artery occlusion, MCAO)模型是临床上广泛应用的方法,传统做法是Longa线栓法。本研究拟对鼠脑MCAO后1h、3h、9h及24h不同时间点的远隔部位进行磁共振ADC的连续检测,并与TTC染色、光学显微镜和和免疫组化法鉴定凋亡细胞情况相对照,评价ADC值在MCAO后不同时间点远隔部位中的应用价值,为运用ADC值评价神经机能联系不能学说提供实验基础和理论依据。材料和方法：参照Longa法建立MCAO大鼠模型,并加以改进。实验动物均在制模后1h、3h、9h及24h各时间点行MRI动态观察。使用美国GE公司3.0 T超导高场磁共振扫描仪,行横断面T1加权成像(T1-weighted imaging, T1 WI)、T2加权成像(T2-weighted imaging, T2WI)和DWI序列成像检查。参照MRI扫描层面,取各组时间点动物2只,将鼠脑切成3mm厚的脑组织切片,红四氮(2,3,5-triphengltetrazolium chloride, TTC)染色后,观察脑组织颜色变化；参照MRI扫描层面,取各组时间点动物2只,对脑组织切片取材,分别进行HE染色光镜检查和免疫组化法鉴定凋亡细胞。结果：1. MCAO后随梗死时间延长,梗死后1～24h DWI上,梗死核心面积逐渐变大,信号增高,邻近皮层逐渐累及,24h信号变化趋于稳定,ADC图上,梗死区面积及信号随时间逐渐变化。MCAO后梗死核心不同时间点ADC值下降的程度不同,1h ADC值较正常对照组明显下降,随时间延长逐渐升高,但仍未恢复正常值水平。配对样本t检验显示,大鼠MCAO后,梗死核心区在不同时间点与正常对照组大鼠相应部位相比,均明显降低(P<0.01)。2.鼠脑MCAO后,两侧丘脑ADC值变化情况较为类似,MCAO后1h下降最为明显,左、右侧丘脑较正常对照组分别下降约28%、24%；1～3h迅速回升,3-24h变化较为平稳,但于9h出现轻度下降。MCAO后两侧小脑ADC值变化与丘脑趋势类似。配对t检验显示,正常对照组各对应部位的ADC值无统计学意义(P>0.05)；左侧MCAO后,各部位的ADC值在不同时间点与正常对照组大鼠相应部位相比,均明显降低(P<0.05),特别是MCAO后1h最为明显。3.正常对照组鼠脑组织TTC染色表现为组织红染；实验组DWI梗死区不被染色呈白色；随梗死时间延长,3-24h梗死面积逐渐变大,邻近皮层逐渐累及,但两侧丘脑、小脑各时间点TTC染色均表现为组织红染。4.光镜下正常对照组及实验组大鼠两侧丘脑、小脑神经元核大而圆且位于细胞中央,细胞浆丰富,未见明显异常。大鼠MCAO后1h,光镜下梗死区神经元细胞未见异常,个别神经元细胞显示核深染、胞浆嗜酸性变；3h脑梗死区域少数神经元细胞呈现核深染、胞浆嗜酸性变；MCAO后9h,左侧大脑发生核固缩的神经元细胞数目进一步增多；MCAO后24h,脑梗死区域可见较多核固缩、呈缺血性改变的神经元,部分区神经元细胞核消失,神经元数量减少。但1～24h实验组大鼠两侧丘脑、小脑光镜下神经元仍未见异常。5.随着梗死时间的延长,梗死核心区凋亡细胞的数目也在不断增加,由MCAO后1h的2.400±0.548,逐渐升高至MCAO后24 h的61.000±7.483,其中MCAO后24 h凋亡细胞的数目最多(p<0.05),而丘脑与小脑在各观察时间点均未见凋亡细胞的产生。结论：1.改良MCAO模型操作简单,24h内实验大鼠死亡率低,可重复性好,是ADC值有效量化的理想模型。2.与正常对照组大鼠左、右两侧相比,MCAO后鼠脑远隔部位(如丘脑及小脑)梗死患侧及对侧的ADC值均有统计学差异,说明远隔部位两侧均存在神经机能联系不能现象。3.远隔部位(如丘脑及小脑)ADC值随幕上梗死核心变化而变化,间接说明神经机能联系不能现象与神经传导通路关系密切。关键词：缺血性脑梗死磁共振成像表观弥散系数第二部分表观弥散系数评价幕上单侧超急性期脑梗死患者远隔部位的继发性变化背景和目的：缺血性脑梗死后远隔区域的继发性变化,可用神经机能联系不能学说进行解释。它是指局灶的脑组织损害可以导致结构正常的远隔区域出现短暂的功能抑制,且原发损害部位与远隔区域之间存在解剖上的纤维联系。神经机能联系不能在脑梗死患者的康复过程中起到一定的作用,正确认识这一现象,并清楚其在脑梗死后患者功能恢复过程中所发挥的作用就显得非常必要。以往大多数学者应用单光子发射计算机断层成像术(Single photon emission computedtomography, SPECT)和正电子发射断层成像术(Positron emission tomography, PET)对此现象进行研究,但PET/SPECT设备较为昂贵,扫描具有辐射,费用较为昂贵,操作起来也极为不便等原因,而使其应用受限。磁共振动态磁敏感对比增强灌注加权成像(Dynamic susceptibility contrast-perfusion weighted imaging, DSC-PWI)是一种无创性的评价病变区域异常血流灌注情况的检查技术,可用于评价幕上脑梗死后交叉性小脑神经机能联系不能(Crossed cerebral-cerebellar diaschisis, CCD)现象的发生。本研究拟对“国家十一五支撑计划项目”的17例超急性期脑梗死(≤6h)患者的MRI资料进行回顾性分析,结合本组第一部分对ADC值的研究,采用ADC值量化幕上单侧超急性期脑梗死患者远隔部位的变化,并与DSC-PWI灌注参数值进行一致性分析；从ADC图的角度观察幕上单侧脑梗死后远隔区域的信号变化,与DSC-PWI参数图进行比较,从而为临床评估超急性期脑梗死后神经机能联系不能的发生寻求新的方法。材料和方法：2007年12月至2010年9月期间,连续筛选我院经急诊绿色通道诊断的,我院参与“国家十一五支撑计划项目”的17例超急性期脑梗死(≤6h)的患者资料。所有研究对象均使用3.0T超导MR机(GE Medical System)进行头部扫描,采用8通道线圈,均采用DWI及DSC-PWI检查。将MR灌注图像传送到工作站(ADW 4.3),应用GE公司专用灌注软件处理并获得对比剂时间浓度曲线。结果：1.正常成人脑组织同一年龄不同部位的ADC值不同,其中以额叶灰质最高,其次为壳核、额叶白质与丘脑,小脑最低。同一部位ADC值随着年龄的增长而增加。两侧脑组织不同部位的左、右两侧间的ADC值作配对t检验,无明显统计学差异(p>0.05)。超急性期脑梗死患者患、对两侧丘脑相比,ADC值有统计学差异(p=0.032),其他部位没有统计学差异(p>0.05)。超急性期脑梗死患者两侧小脑ADC值均有不同程度下降,配对样本t检验显示没有差异(p>0.05),而梗死对侧小脑与正常组相比,独立样本t检验显示有统计学差异(P<0.05),梗死患侧小脑与正常组相比,没有统计学差异(p>0.05)2.10例超急性脑梗死患者远隔小脑磁共振DSC-PWI及DWI检查中,NEI图上,4例患者有明显灌注缺损,2例患者有轻度灌注缺损,4例患者无灌注缺损。MTE图上,2例患者有明显灌注伪彩差异,2例患者有轻度灌注伪彩差异,2例患者无灌注伪彩差异。ADC图上,3例患者存在明显伪彩差异,3例患者存在轻度伪彩差异,4例患者没有伪彩差异。3.10例超急性脑梗死患者,梗死同侧与对侧小脑相比,NEI测量值相比有统计学差异(p<0.05),10例患者两侧小脑MTE测量值没有明显统计学差异(p>0.05)。结论：1. ADC值可超早期(≤6h)量化幕上单侧脑梗死患者远隔部位神经机能联系不能现象。2.从ADC图的角度观察幕上单侧脑梗死后小脑的信号变化,与磁共振灌注负性增强积分(NEI)参数图有较好的一致性。3.在关注超急性期脑梗死患者梗死部位的同时,也要关注远隔部位的变化。更多还原

【Abstract】 Part I Changes of apparent diffusion coefficient at distant parts in rats after MCAOBackground and Purpose:Ischemic cerebrovascular disease (ICVD) has been seriously threatening human health and quality of life. According to the latest epidemiological survey, the incidence rate of ICVD was 116/10 million,81/10 million for the mortality rate,3%o million for the prevalence rate, and has showed an obvious upward trend in our country. ICVD has been seriously threatening human health and quality of life, so it is important to diagnose and treat this disease earlier. ICVD could cause inhibition of distant areas (such as the thalamus and cerebellum), whose mechanism is still unclear, but may be relevant to diaschisis. In recent years, with the development of the magnetic resonance imaging (MRI) new techniques, especially the diffusion weighted imaging (DWI) techniques, MRI has become more and more sensitive and differential to diagnose ICVD. However, previous scholars put particular emphasis on the evaluation by apparent diffusion coefficient (ADC) values of ICVD lesions itself and ischemic penumbra (IP). For the research of secondary changes in distant regions after supratentorial ischemic stroke, there has not yet related reports at home and abroad. In order to study human cerebral infarction better, it is important to establish a cerebral ischemic model in rats with reproducible, strong stability and cerebrovascular characteristics similar to human. Intraluminal suture method is widely used to establish a middle cerebral artery occlusion (MCAO) model in clinical, and the traditional approach is Longa suture method. In this study, remote undamaged regions of rats brain after MCAO were detected continuously by MRI ADC at 1h,3h, 9h and 24h for different time points, compared with TTC staining, optical microscopy and immunohistochemical identification of apoptotic cells, to evaluate the evaluation by ADC values after MCAO at different time points in the remote regions, providing experimental and theoretical basis on the evaluation of diaschisis by ADC values.Materials and Methods:The MCAO model of rats was established based on Lon-ga method, having been modified. The experimental group of rats were dynamically examined by magnetic resonance imaging (MRI) at 1h,3h,9h, and24h after MCAO. With high-field 3.0 T superconducting magnetic resonance scanner of GE in U.S., the line cross-sectional T1-weighted imaging (TIWI), T2-weighted imaging (T2WI) and DWI imaging sequence were performed. For the sake of 2,3,5-triphengltetrazolium chloride (TTC) staining, the brain of two rats in each group were cut into slices every 3 mm, obtained correspondently to MRI scans, to observe that whether brain tissue samples were dyeing or not.2 specimens in each time point which were obtained correspondently to MRI scans, were taken into cut brain tissue, to be observed by microscopy after HE staining and identified apoptotic cells by immunohistochemical respectively.Results:1. With the time coursing, the infarct core size and signal intensity became larger, involving the adjacent cortex gradually during 1h-24h after MCAO in the image of DWI, and the signal change became gradually stable during at 24h after MCAO. The infarct area and the signal were gradually changed with time in the map of ADC. The decreased levels of ADC values in the core of infarction were not consistent with each inspection time after MCAO. ADC values decreased significantly at 1h compared with normal control group, and increased gradually with time, but had not yet resumed normal levels. Paired sample t test showed that, ADC values of the infarction core at each inspection time were significantly lower after MCAO compared with the corresponding parts of the control group (P＜0.01).2. After MCAO, ADC values varied similarly on bilateral hypothalamus, it decreased the most obviously at 1h after MCAO, on the left and right thalamus it decreased by about 28%,24%separately compared with normal control group; During lh-3h after MCAO it rebounded, During 3h-24h after MCAO it varied more steadily, but mildly decreased at 9h after MCAO. The change of ADC values on both sides of the cerebellum and thalamus showed a similar trend after MCAO. Paired t test showed that in normal control group, the difference of ADC values between the corresponding parts was not significant (P>0.05); after MCAO on the left, compared with the normal control group, the ADC values in different parts decreased significantly (P<0.05) at different time points, in particular, the most obviously at 1h after MCAO.3. In the control group, the rat brain showed red staining on TTC. The infarct core showed on DWI in the experimental group was not stained and showed white. With the time extending after MCAO, infarct size increased gradually within 3-24h, gradually involving the adjacent cortex. But both sides of the thalamus and cerebellum showed red staining on TTC in each time point.4. In the normal control group and experimental group, by light microscope, neuronal nucleus of both sides of the thalamus and cerebellum located in the central of cells were large and round and cytoplasm was full, showing no obvious abnormalities. At 1 h after MCAO, neurons of infarct area showed no abnormality by light microscope, but individual nuclear showed anachromasis and cytoplasm showed acidophily alteration. At 3h after MCAO a small number of nucleus of neurons showed anachromasis and cytoplasm showed eosinophilic alteration in infarction area. At 9h after MCAO, the number of neurons of nuclear condensation on the left brain increased in further. At 24h after MCAO, more neurons with pyknosis and ischemic change were seen in the cerebral infarction region, and in some regions nucleus of neurons disappeared and the number of neurons reduced. However, the neurons of both sides of thalamus and cerebellum showed no abnormality by light microscope in 1h-24h experimental groups.5. With the time extending after infarction, the number of apoptotic cells in infarction core gradually increased, from 2.400±0.548 at 1h after MCAO to 61.000±7.483 at 24h after MCAO. At 24h after MCAO the largest number of apoptotic cells was largest (p<0.05), while no apoptotic cell was showed in thalamus and cerebellum at all time points.Conclusion:1. Modified MCAO model is operated simply with low mortality in 24h and good repeatability, and is the ideal model to quantify ADC value effectively.2. Compared with the normal control group, the ADC values of ipsilateral and contralateral remote regions (thalamus and cerebellum) of infarction after MCAO are significantly different. It explains that the phenomenon of diaschisis exists on both sides of the distant parts.3. ADC values of remote regions (thalamus and cerebellum) varies along with the change of the supratentorial infarction core, to a certain extent, it is illustrated indirectly that the phenomenon of diaschisis and neural pathways are closely related. Part II The secondary changes by apparent diffusion coefficient in the distant parts of hyperacute unilateral supratentorial cerebral infarctionBackground and Purpose:Secondary changes in the remote region of ischemic stroke are explained by diaschisis. Diaschisis is the phenomenon that the focal damage of brain tissue could lead to a brief inhibition in remote regions of normal structure, and the primary damage site and the remote regions are contacted with fiber anatomically. Diaschisis played a role in the recovery of cerebral infarction, so it is very necessary to realize the phenomenon correctly and to understand the role it played in the functional recovery of cerebral infarction. Most previous scholars utilized single photon emission computed tomography (SPECT) and positron emission tomography (PET) to study this phenomenon, but the factors such as expensive equipment, scanning with radiation, more costly, inconvenient to operate made its application limited.Dynamic susceptibility contrast-enhanced magnetic resonance perfusion weighted imaging (DSC-PWI), a noninvasive technique making evaluation of abnormal perfusion lesions, can be used to evaluate the phenomenon of crossed cerebral-cerebellar diaschisis (CCD) after supratentorial cerebral infarction. In this study, MRI datas of 17 patients of hyperacute cerebral infarction (≤6h) are retrospectively analyzed, who are from our hospital in the "Project supported by National Eleventh Five-Year Plan". Combined with the ADC values study in first part of this group, ADC value is utilized to quantify the change in the remote parts of hyperacute unilateral supratentorial cerebral infarction, and the consistency analysis of it and DSC-PWI perfusion parameters is made simultaneously. The signal change in the remote regions of unilateral supratentorial cerebral infarction is observed from the ADC map perspective, and is compared with DSC-PWI parameter map, thereby a new evaluation method for clinical assessment of diaschisis secondary to hyperacute cerebral infarction was provided. Materials and Methods:From December 2007 to September 2010, all data of 17 patients of hyperacute cerebral infarction (≤6h) diagnosed by our hospital emergency room were continuously selected.17 patients were from our hospital in the "Project supported by National Eleventh Five-Year Plan". All of the subjects were undertaken head scan using the 3.0 T superconducting MR unit (GE Medical System),8-channel coil, DWI and DSC-PWI examination. DSC-PWI were delivered to the workstation (ADW 4.3), contrast time intensity curve was got after the handle of special perfusion software of GE.Results:1. In the same age group, ADC values varied in different parts of brain tissue in normal adult, among which the frontal gray matter valued the highest, followed by the frontal white matter and thalamus. In the same part, ADC values increased along with age increasing except the cerebellum. Paired t test showed that there was no significant statistical difference for ADC values of both sides of the brain tissue in different parts (p>0.05). In patients with hyperacute cerebral infarction, ADC values of the both sides of the thalamus were compared, and there was significantly different (p=0.032), however, in other region the different of ADC values were not significantly (p>0.05). ADC values of both sides of the cerebellum in patients with hyperacute cerebral infarction were decreased to different levels and paired sample test showed no difference (p>0.05). Compared with the normal group, ADC values of cerebellum contralateral to infarction were statistically significant different in independent samples t test (P<0.05). Compared with the normal group, ADC values of cerebellum ipsilateral to infarction were not significantly different (p>0.05).2.10 cases of hyperacute cerebral infarction were undertaken DSC-PWI and DWI MRI examination in distant cerebellum. On NEI map,4 patients showed significant perfusion defects,2 patients showed mild perfusion defect, and 4 patients showed no perfusion defects. On MTE Fig,2 patients showed significant perfusion differences in pseudo-color, 2 patients showed mild perfusion differences in pseudo-color,2 patients showed no perfusion pseudo color difference. On ADC map,3 patients showed significant differences in pseudo-color,3 patients showed slight differences in pseudo-color,4 patients showed no pseudo color difference.3. In 10 cases of hyperacute cerebral infarction, there was significantly different (p<0.05) in NEI measured values between ipsilateral and contralateral cerebellum of infarction, while there was no statistical difference (p> 0.05) in MTE measured values between bilateral cerebellum.Conclusion:1. ADC values can quantify diaschisis in the distant parts of hyperacute (≤6h) unilateral supratentorial cerebral infarction.2. ADC maps can get a good consistency with enhanced magnetic resonance perfusion negative points (NEI) parametric maps.3. The infarction itself should be paid attention, and its distant part should also be concern about.更多还原