【作者】 宋峰；

【导师】 宦怡；

【作者基本信息】 第四军医大学 ， 影像医学与核医学， 2008， 博士

【摘要】 研究背景:多发性硬化（multiple sclerosis,MS）是一种常见的中枢神经系统（central nerves system, CNS）的炎性脱髓鞘疾病。临床上以病变部位及发病次数的多发性为特点。病理上以中枢神经系统中局灶性脱髓鞘和不同程度的炎性病变﹑轴突损伤和胶质瘢痕的形成为特征。虽然多发性硬化的标志性病理改变是脱髓鞘,然而最近的研究表明轴突的病理改变在疾病的早期阶段业已存在。脊髓病变在多发性硬化患者中广泛存在,而且研究表明因轴突丧失而引起的脊髓萎缩与多发性硬化患者永久性神经功能障碍密切相关。迄今为止,人们对于轴突损伤的病理机制知之甚少,且没有任何一种有效的治疗能够阻止轴突的损伤。因而寻找一种新的有效手段用于研究轴突损伤的潜在病理机制并对治疗的有效性进行精确的监测是迫切而必要的。核磁共振（Magnetic Resonance, MR）技术广泛用于多发性硬化的研究,然而传统的MR技术并不具有病理特异性。近年来诸如弥散张量（diffusion tensor imaging ,DTI）、氢质子磁共振波谱（1H-MR spectroscopy,1H-MRS）、磁化传递成像（Magnetization Transfer Imaging, MTI）等等一些具有相对病理特异性的MR新技术被用于MS病理机制的研究。特别是DTI技术能有效检测轴突损伤并与脱髓鞘病变相区别。为MS病理机制的研究开辟了新的途径。对人体而言,活捡和尸检是研究MS病理机制的两种有效途径。然而由于只能获得MS病程中的间断性数据,这些数据的可靠性和完整性难免受到一定的限制。因而建立有效的MS动物模型是必要的。实验性自身免疫性脑脊髓炎（experimental allergic encephalomyelitis, EAE）是一种自身免疫性神经系统疾病,被认为是MS的首选动物模型。人们对MS病理机制及治疗的认识部分来源于EAE模型。啮齿类动物被广泛应用于EAE模型的建立。遗憾的是,由于啮齿类动物较小的中枢神经系统尤其是脊髓体积,其EAE模型并不适合于核磁共振研究。即使使用高场微型磁共振系统（high-field magnetic resonance microimaging scanner）,许多新技术的应用仍然受到一定限制。少量研究使用灵长类动物模型进行MR研究,然而由于灵长类动物获取困难且价格昂贵,因而难以进行大规模研究。最近的研究表明,猪是一种较适合建立EAE模型的大型动物。研究目的:1.（1）建立可靠的适合MRI成像的猪EAE模型。（2）对EAE模型脊髓病变的病理机制进行研究。2.（1）评估3T临床MRI系统用于猪脊髓轴位DTI成像和定量检测的可行性。（2）定量分析DTI参数﹑脊髓病理改变和EAE临床分数的相关性。（3）评估DTI参数监测EAE发病及疾病进展的能力。材料与方法:1.应用牛脊髓匀浆（ spinal cord homogenate ,SCH）、福氏佐剂和结核杆菌免疫雌性45-50kg的商品猪,耳静脉注射百日咳杆菌,诱发猪EAE动物模型。2.应用组织病理染色和免疫组化技术研究EAE的病理机制,具体包括:苏木素-伊红（Hematoxylin-Eosin staining, HE）染色显示炎性浸润,LFB（Luxol fast blue）染色显示脱髓鞘病变,Glees银染显示轴突丧失,淀粉样前体蛋白（Myloid precursor protein, APP）免疫组织化学染色显示急性轴突损伤。3. 5头正常雌性45-50kg的商品猪用于评估3T临床MRI系统进行猪脊髓DTI成像和定量检测的可行性。3T临床MRI系统对猪脊髓进行常规T1WI、T2WI及DTI成像,DTI扫描采用基于平行采集技术（Integrated Parallel acquisition techniques , iPAT ）的平面回波（ Echo Plane Imaging,EPI）脉冲序列。定量测量C2/C3平面脊髓轴位白质四个不同感兴趣区（前柱,左侧柱,右侧柱和后柱）的轴向弥散（Axial diffusivity, AD ）、径向弥散（radial diffusivity ,RD）、部分各向异性（fractional anisotropy,FA）和表观弥散系数（apparent diffusion coefficient ,ADC）值。测量采用西门子公司的Syngo MRB13图像分析软件平台。用Bland–Altman法对DTI各参数的可重复性进行评估,统计软件采用MedCalc V7.5。4. 8头急性猪EAE模型（P1-P8）用于定量分析DTI参数﹑脊髓病理改变的相关性。分别于不同病程阶段（两头在进展期,临床分数分别为1和3;两头在峰值期,临床分数均为5;两头在缓解期,临床分数分别为4和2;两头在完全缓解期,临床分数为0）行MR扫描。于MR扫描后立即处死取脊髓标本分别进行组织病理和免疫组化染色。对DTI和定量病理改变进行相关性分析,统计软件采用SPSS14。5. 2头急性猪EAE模型（EB、EC）用于评估DTI参数监测EAE发病及疾病进展的能力。在EAE疾病的全程,每日评估动物的临床分数,分别在不同的时间点行MRI扫描并定量测量DTI参数值。用Spearman秩相关对DTI和临床分数进行相关性分析,统计软件采用SPSS14。结果:1.通过后腿皮内多点注射牛脊髓匀浆、福氏佐剂和结核杆菌,耳静脉注射百日咳疫苗的方法,成功建立了急性猪EAE模型。病程表现为急性单向完全缓解型。2.急性猪EAE模型的脊髓病理改变以炎性细胞浸润和急性轴突损伤为主。仅在疾病峰值期可见少量脱髓鞘改变。在缓解期可见少量轴突丧失且在临床症状完全缓解后少量轴突丧失持续存在。3.采用基于iPAT技术的EPI脉冲序列,3T临床MRI系统可以获得较高质量的猪脊髓DTI图像。Bland–Altman法评估显示脊髓DTI参数值具有较好的可重复性。AD, RD, FA,和ADC的正常值分别为1.81×10^-3 （±0.06） mm²/s, 0.32×10^-3 （±0.02） mm²/s, 0.72×10^-3 （±0.08）和0.95×10^-3 （±0.11） mm²/s。这些DTI参数值与正常人脊髓的DTI参数值基本相符。4. AD值随急性轴突损伤的增加而下降,两者呈明显负相关（r = -0.84, P <0.001）, FA与急性轴突损伤虽然也有负相关的趋势但相关性不强（r = -0.31, P<0.05）。5. AD与临床分数间表现出很强的相关性（两只动物的相关系数分别为:r=-0.86, P<0.001;r=-0.92, p<0.001）。结论:1.牛脊髓匀浆能有效诱导急性单向完全缓解型猪EAE模型。静脉注射百日咳疫苗、高剂量结核杆菌、皮内注射可能是促进EAE发病的有利因素。2.急性猪EAE的脊髓病理改变以炎性浸润和急性轴突损伤为主。伴有少量的脱髓鞘病变。急性轴突损伤可能独立于脱髓鞘病变而单独存在。绝大多数急性轴突损伤是可逆的。急性轴突损伤可能是引起猪EAE临床神经功能障碍的主要因素。3.采用基于iPAT技术的EPI脉冲序列,3T临床MRI系统能获得较好的猪脊髓DTI图像且能进行DTI参数的轴位定量测量。猪EAE模型是进行MRI研究的较好动物模型。4. AD值在时间和空间上均能反映脊髓轴突的急性损伤,AD是检测脊髓轴突损伤的特异性参数。5. AD是监测脊髓轴突损伤的敏感参数,AD可能是研究MS和EAE模型轴突损伤的病理机制并且进行轴突损伤治疗效果监测的敏感指标。更多还原

【Abstract】 BackgroundMultiple sclerosis （MS） is a chronic progressive disease of the central nervous system （CNS） which is histopathologically characterized by focal demyelinated lesions with different degrees of inflammation, axonal damage and glial scar formation. Although the hallmark of the process is demyelination, axonal pathology has been known to occur in multiple sclerosis from the earliest post-mortem descriptions of the disease. It has been widely speculated that axonal loss is the pathologic correlate of irreversible neurological impairment in MS. So far, no therapy has been proven to stop or prevent axonal injury, the pathogenesis of which is largely unknown. So a new marker to investigate underlying pathogenesis processes of MS and guide therapeutic management in more precisely is required.In humans, biopsy or autopsy are only approach to investigate underlying pathogenesis processes in MS, The data are of course limited as they give only a snapshot of the disease process .Therefore, animal models of disease are necessary to elucidate the manner in which diffusion properties reflect developmental or pathological changes. Experimental allergic encephalomyelitis （EAE） is a neurological autoimmune disease and the primary animal model for MS. Current understanding of the mechanisms of disease and therapy in multiple sclerosis has been partially derived from the model of EAE.MRI has proven to be a very sensitive technique for the detection of multiple sclerosis （MS） lesions and has provided remarkable insight into the dynamic nature of the disease process. However, our understanding of the relationship of these imaging findings to changes occurring in the tissue is rudimentary, and very few formal studies have addressed this issue. Diffusion tensor imaging （DTI） takes advantage of the anisotropic nature of water diffusion in biological samples to extract detailed micro-structural information. This technique has been widely applied for the evaluation of CNS development and pathology. A recent study indicated that directional diffusivities may serve as surrogate markers of axonal and myelin degeneration in EAE-affected mouse.The spinal cord is a common site of pathology in multiple sclerosis and is a significant contributor to clinically disability. Most MRI studies of EAE were performed on small animals such as rat or mice. The small size of the spinal cord and the presence of motion artefacts, due to cerebrospinal fluid and cardiac pulsation as well as respiration are challenging for MRI study in vivo. Up to now, only a few studies were performed on high-field magnetic resonance microimaging scanner. In view of the relatively low ratio between the RF coil and the swine cord and the recently comment that it would be beneficial to develop and characterize the EAE model in large animals. We select swine EAE model in our study. Recently high magnetic field strength （3T） clinical scanner is used. The larger core than microimaging scanner and high resolution may be a precondition to perform DTI study on spinal cord of EAE model in large animals in vivo.Objectives1. （1） Induction of Experimental Allergic Encephalomyelitis in Swine.（2） To study the spinal cord pathological changes of acute experimental allergic encephalomyelitis in swine.2. （1） To evaluate the feasibility and reproducibility of in vivo diffusion tensor imaging （DTI） of the swine spinal cord by 3T clinical scanner system.（2） To investigate the quantitative relations between DTI parameters and axonal injury pathological staining and clinical score.（3） To evaluate the sensitivity and the ability of DTI parameters to monitor axonal injury through the course of EAE.Materials and methods1. Female commercial swine, having a body weight of about 50–60 kg, were used in this study. The EAE model was induced in these swine by immunization with bovine spinal cord homogenate （SCH）, complete Freund’s adjuvant and M. tuberculosis.2. Neuropathological staining comprised haematoxylin and eosin （HE）, luxol fast blue （LFB）, and Glees silver staining. Immunohistochemical staining was performed using the Envision method. The primary antibody was polyclone anti-amyloid precursor protein （APP）. LFB stains myelin, APP immunostaining demonstrate acute axonal damage and Glees silver staining detect axonal lose.3. Five control swine were imaged twice using a 3 Tesla clinical MRI unit, 6 days apart, to determine inter-assay variability. Quantitative analysis of DTI data were performed on a Syngo MRB13 imaging software platform. DTI metrics were measured in each axial section in specific ROIs placed in spinal cord at the anterior, right lateral, left lateral and posterior regions of the spinal cord at the C2/C3 vertebral level.4. Eight EAE swine （P1-P8） were used to investigate the correlation between DTI parameters and quantitative axonal injury pathological staining, Swine were performed MRI studies at distinct clinical phases and sacrificed just after the MRI study. Then spinal cords were dissected and fixed overnight in 4% paraformaldehyde for neuropathological and immunohistochemical staining. Correlations between direction diffusivity and quantitative immunohisto -chemistry staining were explored with Spearman liner correlation.5. Two EAE swine （EB, EC） were used to monitor the acute EAE onset and progression. MRI studies were set to start at 6 days after immunization. Subsequently, examinations were relatively equally spaced and in later stages adjusted to disease development. Correlations between direction diffusivity and clinical score were explored with Spearman rank correlation.Results1. Experimental allergic encephalomyelitis （EAE） was inoculated with bovin spinal cord homogenate （SCH） in the swine to create a large animal model of multiple sclerosis. The clinical course was primarily monophasic acute course with complete remission and recovery. Variables which appeared to foster disease were pertussis toxin, higher concentrations of M.tuberculosis, and intradermal inoculations.2. In our acute monophasic swine EAE model, the main histopathological changes were infiltration of inflammatory and acute axonal injury. Only a few of minor areas of demyelination and axonal lose were found at the peak point of the clinical attack. APP positive axonal and demyelination could no longer be detected by the time of clinical disease recovery completely. The numbers of axonal lose was stabile.3. All DTI parameters were compared between two measurements conducted 6 days apart. There were no statistically different at the 95% confidence level. The quantitative DTI parameters from each different region of spinal cord were not statistically different. The AD, RD, FA, and ADC were respectively equal to 1.81×10^-3 （SD = 0.06） mm²/s, 0.32×10^-3 （SD = 0.02） mm²/s, 0.72×10^-3 （SD = 0.08） and 0.95×10^-3 （SD = 0.11） mm²/s. These results were in accordance with previous studies in human spinal cord.4. The relationship between axial diffusivity and acute axonal injury was quite strong, with increasing acute axonal injury, axial diffusivity decreased （Spearman r=-0.84, P <.001）. FA correlated less well with axonal injury （r=-0.31, P<0.05）.5. Clear correlations between clinical disease and axial diffusivity was found in all two EAE swine respectively （r=-0.86, P<0.001 and r=-0.92, p<0.001）.Conclusions1. Intradermal inoculations of bovin spinal cord homogenate was a feasibility way to inoculate acute monophasic and complete remission experimental allergic encephalomyelitis （EAE） in the swine.2. In our acute monophasic swine EAE model, the main histopathological changs were infiltration of inflammatory and acute axonal injury. Acute axonal damage may be independent of demyelination, and it’s pathogensis may be different from demyelination. Our data further suggest that in an inflammatory condition like that in MS, demyelination is not a prerequisite for axonal injury.3. High quality DTI of spinal cord could be acquired by using iPAT-based echo-planar imaging technique. It is feasibility to measure DTI parameters in the axial plane of the swine spinal cord in vivo by 3T clinical scanner.4. The relationship between axial diffusivity and acute axonal injury was quite strong, with increasing acute axonal injury, axial diffusivity decreased.5. AD is a sensitive mark to monitor acute axonal injury in the course of EAE onset and progression and DTI may be a useful noninvasively tool to investigate underlying pathogenesis processes of MS and monitor the effect of experimental treatment for axonal injury.更多还原