【作者】 徐惠；

【导师】 李传福；

【作者基本信息】 山东大学 ， 影像医学与核医学， 2010， 博士

【摘要】 目的：近年来,磁共振成像(magnetic resonance imaging, MRI)技术得到飞速发展,其中,磁共振波谱成像(magnetic resonance spectroscopy, MRS)和功能磁共振成像(functional magnetic resonance imaging, fMRI)使MRI从单纯显示解剖结构扩展到了分析脑组织化合物以及揭示脑组织功能的范畴。1.MRS是利用特殊成像序列检测组织内部分化合物的含量的磁共振技术。近年来人们对人类正常和病理状态下的MRS做了很多研究,取得了丰富的成果,并且将MRS应用于大鼠、新生猪、兔等动物模型进行研究。猴等非人灵长类动物与人类在组织结构、生理、生物化学方面有诸多相似,对其进行生理功能、生化结构、病理生理等方面的研究对认识人类的生理及病理过程有重要意义。然而目前国内外对非人灵长类动物进行MRS的研究相对较少。本课题的研究内容之一就是研究MRS在正常非人灵长类动物(食蟹猴)实验研究中的应用,为将来病理状态下的MRS研究提供对照。2. fMRI自20世纪90年代出现以来,在认知科学研究方面发挥了巨大的作用。人们利用fMRI做了很多中枢神经系统功能定位的研究,如：肢体运动、感觉刺激、情感刺激、学习记忆任务和药物反应试验,都检测到了相应中枢神经系统的活动,并得出了非常有意义的结论。针灸(又称针刺,acupuncture)是中国传统医学的宝贵遗产之一,经过数千年的实践证明是一种重要治疗手段,但是其原理却一直无法以现代医学加以阐明。近年来,fMRI技术的广泛应用,为针灸机制的探讨提供了有利的手段。国内外均有学者应用fMRI直观地观测到针刺引起的中枢神经系统的活动,但是目前为止,尚未见在非人灵长类动物身上进行针灸fMRI的研究。本课题对食蟹猴进行针刺fMRI研究,探讨针刺所引起的脑功能活动的变化。3.嗅觉(olfaction)是人体重要的感觉功能之一,多种现代医学技术被用于嗅觉研究,目前已对嗅觉的机制,包括嗅觉通路、脑区中枢定位、分子生物学机制,以及引起嗅觉障碍的各种因素有了一定的认识。以人为对象通过fMRI研究嗅觉的报道已不少,但是对于非人灵长类动物嗅觉的fMRI未见有相关报道。本课题对食蟹猴在气味刺激的同时,检测嗅觉引起的fMRI变化,探讨嗅觉有关的脑功能活动。方法：1.实验对象及设备食蟹猴(macaca fascicularis),属于灵长目猴科,猕猴属。选取健康食蟹猴六只,均为雄性,年龄4-5岁,采用GE公司产Signa ExciteHD 3.0 T MR扫描仪,正交膝关节线圈,动物麻醉后进行磁共振扫描。2.MRS实验方法及统计学处理MRS采用PROBE/SV扫描,点解析波谱成像序列,分别选取纹状体、额叶、丘脑、小脑为感兴趣区(region of interest, ROI),ROI大小15mm×15mm×10mm,尽量避开颅骨和脑脊液,ROI周围从上、下、左、右、前、后六个方向均进行饱和抑制。每个部位扫描3次。通过MR系统测量各ROI的N-天门冬氨酸/肌酸(NAA/Cr)、肌酸/胆碱(Cho/Cr)比值。运用SPSS13.0软件进行数据分析,采用方差分析对数据进行两两比较,p<0.05为差异有统计学意义。3.针刺fMRI实验设计功能成像扫描过程包括左侧足三里穴(ST36)、右侧足三里穴、左侧足三里穴旁非穴位区针刺功能成像扫描。针刺刺激采用"OFF-ON-OFF"组块设计,即“静息-刺激-静息”设计模式。功能成像BOLD序列共采集128 phase,其中第1～8 phase为静息状态不予针刺；之后以每10 phase为一个组块,分别予以“针刺—静息—针刺—静息”,共采集100phase。针刺任务过程包括：刺入—捻转—拔出。静息状态时银针不留置体内。针刺过程由同一名有10年针刺经验的针灸医师完成。取穴时按照针灸动物实验的常规,根据猴与人体型大小,按比例在食蟹猴体表选取相同部位。针刺手法采用“平补平泻”法,捻转均匀，提插幅度适中。4.嗅觉fMRI实验设计每只实验猴扫描过程包括酒精、醋酸分别刺激下的功能成像扫描。嗅觉刺激采用"OFF-ON-OFF"组块设计,即“静息-刺激-静息”设计模式。刺激方式为予以实验气体吸入,进行实验气味刺激,静息方式为予以空气吸入,无实验气味刺激。功能成像BOLD序列共采集108 phase,其中第1-8 phase为静息状态不予实验气体气味刺激；之后以每10 phase为一个组块,分别予以“刺激—静息—刺激”,共采集100 phase。全部气味刺激过程由同二人合作完成。“刺激”时捏压球囊,使猴吸入酒精或醋酸与空气的混合气体,含水量稍高,有气味刺激；“静息”时使猴吸入含水量稍高、仅含空气的气体,无气味刺激。球囊捏压频率约120次/min,气体流量约1200mL/min。实验全程捏压频率、幅度均一致。5. fMRI扫描参数扫描序列包括FLAIR T1WI序列和fMRI-BOLD扫描。首先使用T1 FLAIR序列进行轴位T1WI扫描,扫描线平行于前后连合连线(AC-PC线),扫描范围自顶部皮肤至延髓下端,包括全部脑组织。T1 FLAIR扫描图像作为fMRI扫描的定位像。fMRI-BOLD扫描采用梯度回波—回波平面成像(gradient echo-echo planar imaging, GRE-EPI)序列,血氧水平依赖(blood oxygenation level dependent, BOLD)法。整个猴脑共扫描16层,128 phase,得到2048幅图像。6. fMRI数据处理采用运行于Matlab (7.0.1)平台的专业医学图像处理软件—统计参数图2(statistical parametric mapping 2, SPM2)处理实验数据。猴脑fMRI处理过程包括以下10个方面：删除前8 phase静息态采集图像、转换图像格式、头动校正、去头皮、图像标准化、fMRI设计、数据调入、评估、计算结果、解剖图像覆盖。数据的评估和计算采用随机效应模式(random effect mode)进行组分析。采用单样本T检验来分析特定任务刺激下组激活的状态。采用两样本T检验来分析比较两种不同任务刺激下组激活的差异,包括针刺左侧足三里穴与右侧足三里穴激活的不同；针刺左侧足三里穴与左侧非穴位区激活的不同；酒精气味与醋酸气味激活的不同。设定检验标准p<0.05,激活区滤过的标准为<10个体素。结果：1.MRS检测结果食蟹猴不同部位脑组织的NAA/Cr比值从高到低依次为：丘脑、小脑、额叶、纹状体,其中,丘脑与其他三类组织均有统计学差异(p<0.05),小脑与纹状体之间有统计学差异(p<0.05)。Cho/Cr比值从高到低依次为：额叶、小脑、丘脑、纹状体,其中,纹状体与额叶、纹状体与小脑之间有统计学差异(p<0.05)。2.针刺fMRI成像结果针刺左侧足三里穴可以观察到在针刺刺激时,左侧中央前回、右侧中央后回、双侧岛叶、楔前叶、右侧颞叶激活,信号增高,其中,激活最强点位于右侧颞上回；额叶、顶叶、枕叶、扣带回散在多处负激活点,在针刺刺激时信号减低,其中,激活最强点位于右侧中央前回。针刺右侧足三里穴可以观察到在针刺刺激时,双侧岛叶、双侧中央后回、右侧中央前回、丘脑、颞叶及小脑激活,信号增高,其中,激活最强点位于右侧颞上回；双侧额叶、顶叶、枕叶、扣带回散在大量负激活点,在针刺刺激时信号减低,尤其以额叶、顶叶居多,其中,激活最强点位于右侧扣带回。针刺左侧足三里穴外侧非穴位处可以观察到在针刺刺激时,右侧中央后回、双侧颞上回、双侧楔前叶激活,信号增高,其中,激活最强点位于右侧中央后回；双侧扣带回、左侧额上回、中央后回、右侧豆状核、桥脑、中脑散在负激活点,在针刺刺激时信号减低,其中,激活最强点位于右侧豆状核。对针刺左侧足三里穴与右侧足三里穴的正激活进行两样本组分析T检验,可以发现针刺左侧比右侧在双侧岛叶、右侧颞叶、右侧中央后回、左侧中央前回、双侧楔前叶、右侧豆状核、右侧丘脑、中脑激活强,其中,激活最强点位于右侧中央后回下部；针刺右侧比左侧在双侧额叶、顶叶、扣带回颞叶等处有多处散在较强激活,其中,激活最强点位于左侧额叶。对针刺左侧足三里穴与右侧足三里穴的负激活进行两样本组分析T检验,可以发现针刺左侧比右侧在双侧额叶、顶叶、颞叶、右侧丘脑、左侧豆状核的负激活强,其中,激活最强点位于右侧尾状核旁白质；针刺右侧比左侧在双侧中央前回、中央后回、双侧丘脑、豆状核、岛叶、楔前叶、右侧颞叶等处有多数散在较强激活,其中,激活最强点位于右侧颞上回。针刺左侧足三里穴与左侧足三里穴旁非穴位区的正激活进行两样本组分析T检验,可以发现针刺穴位比非穴位区在左侧中央前回、双侧楔前叶、右侧颞叶、豆状核、丘脑、胼胝体激活强,其中,激活最强点位于右侧楔前叶,针刺左侧足三里穴旁非穴位区比左侧足三里穴在顶叶、额叶、右侧颞叶、左侧楔前叶等处有散在较强激活,其中,激活最强点位于左侧楔前叶。针刺左侧足三里穴与左侧足三里穴旁非穴位区的负激活进行两样本组分析T检验,可以发现针刺穴位比非穴位区在双侧中央前回、中央后回、左侧额叶、颞叶、楔前叶、右侧丘脑旁激活强,其中,激活最强点位于右侧丘脑旁；针刺左侧足三里穴旁非穴位区比左侧足三里穴在双侧中央前回、楔前叶、右侧豆状核、颞叶、中脑、左侧额叶等处有较强负激活,其中,激活最强点位于右侧楔前叶。3.嗅觉fMRI成像结果嗅觉实验操作时,观察到当使用醋酸、乙醇气味刺激时,食蟹猴的呼吸幅度变浅,呼吸频率减慢,使用空气吸入时,呼吸恢复正常。对食蟹猴行醋酸气味刺激,可以观察到刺激时在双侧中央旁小叶、扣带回、左侧额叶、颞叶、枕叶舌回、丘脑、右侧岛叶、豆状核有激活,激活最强点位于左侧扣带回；负激活点位于扣带回、左侧中央旁小叶、豆状核、舌回,刺激时信号减低,最强点位于右侧扣带回。对食蟹猴行乙醇气味刺激,可以观察到刺激时在双侧中央旁小叶、扣带回、左侧颞叶、额叶、右侧豆状核、中脑、小脑有激活,激活最强点位于右侧扣带回；负激活点位于双侧中央旁小叶、扣带回、双侧中央前回、左侧额叶、双侧尾状核、豆状核、小脑,刺激时信号减低,最强点位于左侧中央前回。对醋酸、乙醇的正激活进行两样本组分析T检验,可以观察到双侧扣带回、右侧丘脑、豆状核中后部区域,醋酸刺激激活信号强于乙醇,最强点位于右侧丘脑；在左侧中央旁小叶、双侧豆状核中前部区域,乙醇刺激激活信号强于醋酸,最强点位于左侧豆状核。对醋酸、乙醇的负激活进行两样本组分析T检验,可以观察到右侧扣带回、右侧豆状核前部区域、左侧豆状核中部区域,醋酸刺激激活信号强于乙醇,最强点位于左侧豆状核；在右侧尾状核、豆状核、左侧丘脑、左侧扣带回,乙醇刺激激活信号强于醋酸,最强点位于右侧尾状核。结论：1.本研究结果表明,可以在临床使用的MRI设备上对非人灵长类动物进行脑MRS的检测研究,其数据反映了脑组织内相应化合物的含量。丘脑NAA/Cr比值大于小脑、额叶、纹状体,小脑NAA/Cr比值大于纹状体。额叶Cho/Cr比值大于纹状体,小脑Cho/Cr比值大于纹状体。非人灵长类动物的MRS检测能准确反映脑组织的结构异同和含量的变化,可以作为生理、病理改变研究的依据之一。2.在麻醉状态下,针刺食蟹猴左侧足三里穴、右侧足三里穴及左侧足三里穴旁非穴位区,fMRI可以显示不同脑组织区域的正激活和负激活。从正激活方面来看,针刺的作用机理可能与中央前回、岛叶及颞上回有较密切关系。针刺足三里穴引起的负激活定位性较差,投射关系分散,引起的效应广泛。扣带回的负激活在针刺机理及针灸止痛中可能有重要作用。针刺穴位与穴位旁非穴位区的激活方式差别很大。3.不同气味刺激引起的激活部位有相似之处,也有区别。海马回钩和眶额回为嗅觉最高级中枢,fMRI可以显示额叶、颞叶的激活。气味刺激引起的中枢反应与中央旁小叶、扣带回、豆状核有密切关系。fMRI检测到的信号变化是综合结果,信号变化不仅包含刺激本身引起的中枢反应,也包括动物对此刺激做出的反应。更多还原

【Abstract】 Objective:Nonhuman primates are similar with humans in the tissue structure and biochemistry. Therefore, the study of their physiological function, biochemistry, pathophysiology and other aspects is important and meaningful in understanding the human physiology and diseases. The studies of magnetic resonance spectroscopy(MRS) and functional magnetic resonance imaging(fMRI) on rats and rabbits have been numerously reported, but this kind of studies on nonhuman primates are relatively rare.1. MRS, based on chemical shift theory, is a noninvasive imaging technique used in studying the normal or pathophysiological changes and making quantitative analysis of metabolites. This study firstly aims to explore the application of MRS in experimental studies on normal macaca fascicularis and to get basic data as a reference for further study of pathological conditions.2. Acupuncture represents one of the oldest and still widely used treatments. The second aim of this study was to detect the fMRI signal changes evoked by acupuncture stimulation on nonhuman primates and to explore the functional localization and its distributing orderliness of the central nervous system(CNS) reactions on acupuncturing the left and right acupoints of Zusanli(ST36) and a false acupoint near the left acupoint of Zusanli respectively, and then to offer reference for future research of both human and nonhuman primates.3. As an important sensory function, olfaction has been studied on the mechanism, such as its transmission, the functional localization and molecular biology. The studies of fMRI evoked by olfaction stimulation on human have been reported, but study on nonhuman primates has not been seen. The third aim of the study is to explore the fMRI signal changes evoked by olfaction stimulation on nonhuman primates.Materials and methods:1. Research objects and MR scanner6 healthy macaca fascicularis, which belong to the genus Macaca, family Cercopithecidae, order Primates, were selected in the study. MR examinations were performed with a quadrature knee joint coil at 3.0T MR scanner, with gradient strength of 40mT/m, gradient switching rate of 150mT/m/ms. All monkeys were anesthetized with ketamine and chloral hydrate before MR scanning.2. Method of MRSProton Brain Exam-Single Voxel(PROBE-SV) using a Point Resolved Spectroscopy Sequence(PRESS) was employed for MRS scanning, and four ROIs were selected in the axial image(the corpus striatum, frontal cortex, thalamus and cerebellum, with the size of 15mm×l5mm×l0mm, avoiding the contamination of the adjacent skull and cerebrospinal fluid). The regions around the ROI were all saturated accordingly. MRS scanning was repeated 3 times. N-acetylaspartate (NAA), creatine (Cr), choline (Cho) and the ratio of NAA/Cr, Cho/Cr were measured respectively. SPSS13.0 software and pairwise comparison were used for data analysis and p<0.05 was considered statistically significant.3. study design for acupuncture fMRIFMRI scannings during acupuncture stimulation at the left acupoint of Zusanli(ST36), the right Zusanli and the area beside the left Zusanli were performed respectively. The block design of the acupuncture stimulation was "rest-stimulation-rest". Each time,128 phases scanning was obtained. No acupuncture stimulation was performed during rest period and acupuncture stimulation was performed during stimulation period. The first 8 phases scanning was in rest period, subsequently every 10 phases scanning was regarded as a block in stimulation or rest period alternately all of that 100 phases. The acupuncture stimulation was performed by an experienced acupuncture physician. The acupoint position was decided based on the proportion of the shapes of human and macaca fascicularis. The acupuncture technique was "pingbupingxie".4. study design for olfaction fMRIFMRI scannings during alcohol and acetum scent stimulation were performed respectively. The block design was "rest-stimulation-rest". The monkey inhaled the air mixed with alcohol or acetum in stimulation period, while in rest period the monkey inhaled pure air.The fMRI protocol was the same as acupuncture fMRI. The stimulations were performed by the same two physicians.5. fMRI scanning parametersFLAIR T1WI and fMRI-BOLD(blood oxygenation level dependent) sequence were performed and 16 slices axial images of the entire brain paralleled to the AC-PC line were obtained. FLAIR T1WI axial images were used as the scout image for BOLD. GRE-EPI sequence was used in BOLD scanning.128 phases scanning were performed and 2048 images were obtained.6. fMRI data analysisData were analyzed by using Statistical Parametric Mapping software-SPM2 implemented in Matlab 7.0.1. The operation process included 10 steps, such as deleting the first 8 phases rest period images, conversion images format, realigning images, removing scalp, normalizing images, fMRI designing and estimating, etc. The outcome was superposed on axial images at last. Through normalizing step, the data were normalized in a standard space template of a macaca fascicularis(Washington University School of Medicine, St. Louis, MO).Random effect mode group analysis was applied in estimating and calculating with one sample t-test for group evoking in a certain stimulation and two sample t-test for the comparison between two stimulations. The test standard p<0.05 and the extent threshold 10 voxels were selected.Results:1. results of MRSNAA/Cr ratios ordering from maximum to minimum were halamus, cerebellum, frontal lobe, corpus striatum. Among them, thalamus and the other three structures were statistically different (p<0.05), the cerebellum and striatum were statistically different (p<0.05). Cho/Cr ratios ordering from maximum to minimum were as follows:frontal lobe, cerebellum, thalamus, striatum. There was significant difference between striatum and frontal cortex, striatum and cerebellum respectively(p<0.05).2. fMRI of acupuncture stimulationAcupuncture stimulation at the left acupoint of Zusanli increased BOLD signal in left precentral gyrus, right postcentral gyrus and temporal lobe, bilateral insula and precuneus. The strongest activation appeared in the right superior temporal gyrus. Deactivation pattern was observed in frontal lobe, parietal lobe, occipital lobe and cingulate gyrus. The obvious deactivation was seen in the right precentral gyrus.The average brain activations evoked by acupuncture stimulation at the right Zuanli were in bilateral insula and postcentral gyrus, right precentral gyrus, thalamus, temporal lobe and cerebellum. The strongest activation was in right superior temporal gyrus. While the deactivations in bilateral frontal lobe, parietal lobe, occipital lobe and cingulate gyrus were observed.Activation pattern of acupuncture stimulation at the nonacupoint beside the left Zusanli was obtained in the right postcentral gyrus, bilateral superior temporal gyri and precuneus. The strongest activation was in the right postcentral gyrus. At the same time, the deactivation pattern was observed in bilateral cingulate gyri, the left prefrontal gyrus and postcentral gyrus, the right lenticular nucleus, pons and midbrain. The obvious signal decrease was observed in the right lenticular nucleus.Two sample t-test between the activations of acupuncture stimulation at the left and the right acupoint of Zusanli was performed. In bilateral insula and precuneus, right temporal lobe and postcentral gyrus, left precentral gyrus, right lenticular nucleus and thalamus, midbrain, the activation was stronger at left Zusanli stimulation. Whereas, the stronger activation in bilateral frontal lobes, parietal lobes, cingulate gyri and temporal lobes were observed by stimulating the right Zusanl.The difference between the deactivation patterns of acupuncture stimulation at the left acupoint of Zusanli and the right one was observed. Stronger deactivations in right thalamus, bilateral frontal lobes, parietal lobes and temporal lobes were observed during left Zusanli acupuncture stimulation. While stronger deactivations in bilateral prefrontal gyri, postcentral gyri, thalamus, lenticular nucleus, insula, precuneus, and the right temporal lobe were recorded by the right Zusanli stimulation.Through two sample t-test between the activations in acupuncturing the left Zusanli and the nonacupoint beside the left Zusanli, the stronger activations in left precentral gyrus, bilateral precuneus, right temporal lobe, thalamus and lenticular nucleus, corpus callosum were observed by stimulating the left Zusanli. Whereas, the stronger activations in parietal lobe, frontal lobe, right temporal lobe, left precuneus were observed by stimulating the nonacupoint.The difference between the deactivation patterns of acupuncture stimulating at the left Zusanli and the nonacupoint beside the left Zusanli was observed as follows:there were stronger deactivations in bilateral precentral gyri and postcentral gyri, the left frontal lobe, temporal lobe and precuneus, the right thalamus by the left Zusanli stimulation. While stronger deactivations in bilateral precentral lobe, precuneus, right lenticular nucleus and temporal lobe, midbrain and the left frontal lobe were observed by the nonacupoint stimulation.3. results of fMRI by olfaction stimulationDuring olfaction fMRI examination, the respiration depth and frequency of the monkeys decreased evidently in scent stimulation period, while resumed to normal when pure air inhaled.When acetum scent stimulation was given, increased BOLD signal was observed in bilateral paracentral lobules, cingulate gyri, left frontal lobe, temporal lobe and lingual gyrus, thalamus, right insula and lenticular nucleus. Deactivation pattern was observed in cingulate gyrus, left paracentral lobule, lenticular nucleus and lingual gyrus.The average brain activations evoked by alcohol scent stimulation were in bilateral paracentral lobules, cingulate gyri, left temporal lobe and frontal lobe, right lenticular nucleus, midbrain and cerebellum. While the deactivations were observed in bilateral paracentral lobules, cingulate gyri and precentral gyri, the left frontal lobe, bilateral caudate nucleus and lenticular nucleus and cerebellum.Through two sample t-test between the activations of the acetum scent and the alcohol scent, the stronger activations in bilateral cingulate gyri, right thalamus, the posterior part of lenticular nucleus were observed by acetum stimulation. The stronger activations in left paracentral lobule, the bilateral anterior parts of lenticular nucleus were observed by alcohol scent stimulation.The difference between the deactivation patterns of the acetum and the alcohol scent stimulation was observed as follows:there were stronger deactivations in right cingulate gyrus, the anterior part of the right lenticular nucleus, the medial part of the left lenticular nucleus by acetum stimulation. And stronger deactivations in the right caudate nucleus and lenticular nucleus, left thalamus and cingulate gyrus were shown by alcohol stimulation.Conclusion:1. The results show that MRS study of the brain of nonhuman primates is feasible. The NAA/Cr ratio of hypothalamus is higher than that of the cerebellum, frontal lobe and striatum. NAA/Cr ratio of the cerebellum is higher than that of the striatum. The Cho/Cr ratio of frontal lobe is higher than that of the striatum, and the Cho/Cr ratio of cerebellum is higher than that of the striatum. The MRS examination of nonhuman primates can accurately reflect the difference between brain structures and metabolites.2. Different areas in brain of nonhuman primates present activation or deactivation during acupuncture stimulation at the left acupoint of Zusanli, right Zusanli and the nonacupoint area beside the left Zusanli. According to the activation pattern, there might be a compact relation between acupuncture and precentral gyrus, insula and temporal lobe. The acupuncture at acupoint of Zusanli can induce extensive deactivation. And the deactivation of cingulate gyrus maybe presents an important role to understand and explain the mechanism of acupuncture, acupuncture anesthesia and acupuncture analgesia. There is a signifinant difference between the activations of acupuncture acupoint and nonacupoint.3. BOLD activation and deactivation all can be observed by using scent stimulation. Frontal and temporal lobe can activate as the nerve center of olfaction. The central reaction induced by scent stimulation has an affinity with paracentral lobule, cingulate gyrus and lenticular nucleus.更多还原