【作者】 郭慧；

【导师】 张云亭； 李威； 张敬；

【作者基本信息】 天津医科大学 ， 影像医学与核医学， 2009， 博士

【摘要】 第一部分Wistar大鼠血管源性脑水肿模型弥散成像动态演变研究目的：通过分析Wistar大鼠血管源性脑水肿（vasogenic brain edema，VBE）模型MR弥散成像测量参数与脑组织水含量的相关性，得出该模型最佳扫描方案、弥散成像动态演变规律以及通过该方法在体评估脑组织水含量的可能性，确立该模型血清S-100B水平变化方式。对象和方法：通过黄铜棒液氮冷冻法制备Wistar大鼠VBE模型，按时间点分为2小时组，4小时组，6小时组，8小时组，12小时组，24小时组，48小时组，3天组，5天组及7天组及正常对照组共11组，每组保证6只模型制备成功。分别进行b值=1000s／mm²、b值=1400s／mm²、b值=1800s／mm²弥散加权成像（diffusion weighted imaging，DWI），b值=1000s／mm²弥散张量成像（diffusion tensor imaging，DTI），快速自旋回波-反转恢复（fast spin echo-iversion recovery，FSE-IR）T₁加权成像，病灶侧及对照侧分别计算相应表观弥散系数（apparent diffusion coefficient，ADC）值、指数表观弥散系数（exponential apparent diffusion coefficient，eADC）值、平均弥散系数(average diffusion coefficient，DC_avg)值、部分各向异性（fractional anisotropy，FA）值和T₁弛豫时间，灌注取脑，对应层面对应部位取材测量脑组织水含量，取血测量血清S-100B蛋白水平。通过单因素方差分析进行DWI信号噪声比（signal noise ratio，SNR）及各测量参数不同b值间比较，相同b值下各弥散参数、T₁弛豫时间、脑组织水含量、血清S-100B水平各时间点变化比较，并通过pearson相关分析分析各弥散测量参数、血清S-100B水平与脑组织水含量的相关性。结果：①b值=1000s／mm²图像可以获得最佳SNR，不同b值下各时间点SNR呈不同演变趋势，b值=1000s／mm²时由于T₂透射效应作用较易显示病变；②ADC值在各个时间点随b值增加呈下降趋势，eADC值反之；③脑组织水含量随时间变化呈先升后降趋势，于4小时、6小时、8小时间未见统计学差异；④各b值下ADC值、eADC值以及DC_avg值、FA值和T₁值与脑组织水含量显示良好的相关性；其中以b值=1000s／mm²时ADC值与其相关性最高；⑤血清S-100B蛋白水平于24小时组达高峰，与脑组织水含量相关系数较低。结论：①改进黄铜棒液氮冷冻法致Wistar大鼠VBE模型，确定冷冻时间为2分可以达到比较理想的病程及冷冻程度，进行MR弥散成像时去除头皮软组织保持局部干燥是减少图像伪影的关键，通过脑组织水含量及MR弥散成像观察最终确立2小时、6小时、12小时、24小时、48小时、3天、5天和7天8个观察时间点；②确定DWI成像b=1000 s／mm²为最佳b值，同时ADC值与脑组织水含量相关性最高，可以在体对于脑组织水含量进行定量评估；DTI相对于DWI成像时间更长，参数测量与脑组织水含量相关性并未显示出优势；在体评估脑组织水含量而不关心组织各向异性时可选用DWI序列节省检查时间；③T₁值在体测量脑组织水含量研究显示与脑组织水含量存在良好相关性，但成像时间较长，DWIADC值测量更具有相对优越性；④通过血清S-100B测量建立该模型脑组织损伤程度血清学评价指标。第二部分Wistar大鼠血管源性水肿模型血脑屏障通透性动态演变研究目的：通过伊文氏蓝（Evans blue）染料定量分析血脑屏障（blood brain barrier，BBB）通透性，探讨Wistar大鼠VBE模型BBB通透性随时间演变过程，确定Evans blue对弥散成像各测量值的影响。对象和方法：通过黄铜棒液氮冷冻法制备Wistar大鼠VBE模型，按时间点分为2小时组，4小时组，6小时组，8小时组，12小时组，24小时组，48小时组，3天组，5天组及7天组及正常对照组共11组，每组保证6只模型制备成功。在时间点前2小时经股静脉注射Evans blue染料，相应时间点行b=1000s／mm²DWI及DTI，病变侧及对照侧分别计算相应ADC值、eADC值、DC_avg值和FA值，灌注取脑，对应层面对应部位取材测量脑组织水含量及Evans blue含量。通过独立样本t检验分析模型制作可重复性及Evans blue是否会对弥散测量值产生影响。通过单因素方差分析进行脑组织Evans blue含量、脑组织水含量、各弥散参数值各时间点变化比较，并通过pearson相关分析各弥散测量参数与脑组织水含量的相关性。结果：①通过Evans blue注射组与未注射组脑组织水含量比较显示模型制作稳定性良好，Evans blue注射后会造成病变部位ADC值、DC_avg值的降低、eADC值的升高，而对FA值测量无明显影响；各测量值与脑组织水含量相关系数较Evans blue未注射组略减低；②脑组织Evans blue含量在模型制作后2小时即达峰值，后随时间延长缓慢恢复，7天时间组仍高于正常。结论：①通过Evans blue定量分析确立黄铜棒液氮冷冻法致Wistar大鼠VBE模型BBB通透性动态演变过程，与脑组织水含量变化趋势不同，在模型制备2小时后BBB通透性即达峰值，后缓慢恢复；②首次发现Evans blue注入后对VBE病变处弥散成像平均弥散能力测量值产生影响，而对弥散各向异性值未产生明显影响，在其他条件不变的情况下推测可能由于BBB通透性增高Evans blue漏出造成局部水分子结合状态的改变造成：相应弥散测量值与脑组织水含量仍显示较高相关性。第三部分Wistar大鼠血管源性脑水肿模型水通道蛋白-4免疫组织化学染色动态观察研究目的：通过免疫组化方法半定量观察Wistar大鼠VBE模型室管膜细胞水通道蛋白-4（aquaporin-4，AQP-4）表达随时间动态演变，并分析其与MR弥散成像各测量参数的相关性。对象和方法：通过黄铜棒液氮冷冻法制备Wistar大鼠VBE模型，按时间点分为2小时组，4小时组，6小时组，8小时组，12小时组，24小时组，48小时组，3天组，5天组及7天组及正常对照组共11组，每组保证6只模型制备成功。行b=1000s／mm²DWI及DTI检查，病变侧及对照侧计算相应ADC值、eADC值、DC_avg值和FA值，灌注取脑，对应层面石蜡包埋切片行AQP-4免疫组化染色。通过imagepro-plus软件半定量分析室管膜细胞AQP-4表达累积光密度（integrate optical density，IOD）值，确定其动态演变规律及与弥散各参数相关性。结果：①AQP-4表达呈先下调后上调再下调趋势，2小时组室管膜细胞AQP-4表达即出现明显下调，并与6小时组达到最低值，8小时组可见AQP-4表达出现上调，于24小时组达高峰，随后表达水平缓慢下降，于7天组仍高于正常值；②AQP-4表达半定量于弥散各参数相关系数较低，在0.2902～0.4475之间。结论：确立了黄铜棒液氮冷冻法致Wistar大鼠VBE模型室管膜细胞AQP-4表达随时间动态演变规律，呈先下调后上调再下调趋势；该结果为下一步药理学研究奠定了基础。更多还原

【Abstract】 PartⅠA dynamic evolution of MR diffusion imaging in vasogenicbrain edema model of Wistar ratsObjective: To establish the best scan mode and the dynamic evolution mode ofdiffusion imaging in vasogenic brain edema（VBE） model of Wistar rats through theanalysis of the correlations between the parameters of diffusion imaging and watercontent of brain tissue. To evaluate the value of diffusion imaging in quantification ofbrain water content in vivo. To establish the dynamic evolution mode of S-100B inserum. Materials and Methods: VBE model of Wistar rats was made by cold injurywith copper rod which cooled with liquid nitrogen. There were 11 groups（normalcontrol, 2hrs, 4hrs, 6hrs, 8hrs, 12hrs, 24hrs, 48hrs, 3d, 5d and 7d）, and each group had6 rats. Diffusion weighted imaging（DWI） with 3 b values （1000s/mm², 1400s/mm²,1800s/mm²）, diffusion tensor imaging（DTI） with b=1000s/mm² and fast spinecho-iversion recovery（FSE-IR） T₁ weighted image were performed, the values ofapparent diffusion coefficient（ADC）, exponential apparent diffusion coefficient（eADC）, average diffusion coefficient(DC_avg), fractional anisotropy（FA） and T₁ werecalculated in the lesion side and contralateral side, the corresponding brain tissueswere gotten to calculate the water content. Blood samplings from venae femoraliswere used to test the level of S-100B in serum. ANONA were used to analysis thedifferences of the signal noise ratio（SNR） of DWI, the parameters of different bvalues and the dynamic evolutions of the parameters of diffusion imaging in the sameb value, T₁ time, water content of brain tissues and the level of S-100B in serum. Thecorrelations between the parameters of diffusion imaging, the level of S-100B inserum and the water content of brain tissue were analyzed with pearson correlationanalyzation. Results:①The SNR was best when b=1000s/mm², and SNR showeddifferent changing mode in different b values, and when b=1000s/mm² the lesionswere more obvious because of T₂ shine through effect;②ADC values were decreased when b values were increased, and eADC values were opposite;③The water contentof brain tissues first increased and then decreased with the time evolution, and therewas no significant difference in groups between 4hrs, 6hrs and 8hrs;④The values ofADC, eADC, DC_avg, FA and T₁ showed good correlations with the water content ofbrain tissue, and the ADC values of b=1000s/mm² had the highest correlationcoefficient;⑤The level of S-100B in serum had the peak value in 24hrs group, andhad a low correlation coefficient with the water content of brain tissue. Conclusion:①2 minutes is the most suitable time for cold injury in this model, and removing thesoft tissues of scalp and keeping the local site dry are the key points to avoid theartifact of the diffusion imagings, 8 groups（2hrs, 6hrs, 12hrs, 24hrs, 48hrs, 3d, 5d and7d） are made sure to observe the VBE evolution;②b=1000s/mm² is the best b value,ADC shows the best correlation with the water content of brain tissue whenb=1000s/mm², and it can be used to quantitative analysis the water content of braintissue in vivo. DTI has a longer scan time compared with DWI and the parameters ofDTI have no advantage of correlations with the water content of brain tissues. We canchoose the DWI for in vivo quantification of the water content of brain tissues whenthe anisotropy of tissue is not the focal point to observe in order to save the scantime;③The T₁ values show the good correlation with the water content of braintissue, but it’s scan time is much longer than DWI, so ADC values of DWI are moresuitable to measure the water content of brain tissue in vivo;④The dynamicevolution mode of S-100B in serum can be the evaluating indicator of the degree ofcerebral tissue injury.PartⅡA dynamic analysis of permeability of blood brain barrier invasogenic brain edema model of Wistar ratsObjective: To analysis the dynamic evolution mode of permeability of bloodbrain barrier in VBE model of Wistar rats through the quantification of Evans blue ofbrain tisse, and to make sure whether the Evans blue has influence on measurement ofparameters of diffusion imaging. Materials and Methods: VBE model of Wistar rats was made by cold injury with copper rod which cooled with liquid nitrogen. Therewere 11 groups（normal control, 2hrs, 4hrs, 6hrs, 8hrs, 12hrs, 24hrs, 48hrs, 3d, 5d and7d）, and each group had 6 rats. Evans blue was injected through venae femoralis 2hrsbefore the time points, DWI and DTI with b=1000s/mm² were performed, the valuesof ADC, eADC, DC_avg and FA were calculated in the lesion side and contralateralside, the corresponding brain tissues were gotten to calculate the water content andthe content of Evans blue. Independent-samples t test was used to analysis therepeatability of the VBE model and to make sure whether the Evans blue hasinfluence on measurement of parameters of diffusion imaging. ANOVA was used toanalysis the dynamic changes of the Evans blue content of brain tissue, water contentof brain tissue, and the parameters of diffusion imaging. The correlations betweenthe parameters of diffusion imaging and the water content of brain tissue wereanalyzed with pearson correlation analyzation. Results:①VBE model showed goodrepeatability throught the analysis of water content of brain tissues, and Evans bluecould make the values of ADC and DC_avg decreased, and the values of eADCincreased. Evans blue had no influence on FA measurement. The correlationcoefficients were lower than the groups which had no injection of Evans blue;②The content of Evans blue of brain tissue got the peak value at 2hrs group, and itdeclined gradually, when 7d group it was still higher than normal level. Conclusion :①The dynamic evolution mode of permeability of blood brain barrier is differentfrom water content of brain tissue, it gets the peak value at 2hrs group, and it declinesgradually, when 7d group it is still higher than normal level;②Evans blue hasinfluence on parameters of average diffusion ability, and has no influence onparameters of diffusion anisotropy. The leakage of Evans blue in the lesion site couldhave influence on the molecular binding situation of water, the parameters ofdiffusion imaging after Evans blue injections also show the good correlation with thewater content of brain tissues.PartⅢA dynamic evolution of AQP-4 immunohistochemistry staining in vasogenic brain edema model of Wistar ratsObjective: To analysis the dynamic evolution mode of aquaporin-4 expression ofependymal cells in VBE model of Wistar rats through the semi-quantification ofimmunohistochemistry staining, and analysis of the correlations between theparameters of diffusion imaging and AQP-4 expression. Materials and Methods:VBE model of Wistar rats was made by cold injury with copper rod which cooledwith liquid nitrogen. There were 11 groups（normal control, 2hrs, 4hrs, 6hrs, 8hrs,12hrs, 24hrs, 48hrs, 3d, 5d and 7d）, and each group had 6 rats. DWI and DTI withb=1000s/mm² were performed, the values of ADC, eADC, DC_avg and FA werecalculated in the lesion side and contralateral side, the corresponding slice of the braintissues were gotten to stain with immunohistochemical technique. Thesemi-quantification of immunohistochemistry staining of AQP-4 was made bysoftware imagepro-plus through the value of integrate optical density（IOD）. ANOVAwas used to analysis the dynamic changes of the AQP-4 expression. The correlationsbetween the parameters of diffusion imaging and AQP-4 expression were analyzedwith pearson correlation analyzation. Results:①The expression of AQP-4 showed atrend of first down-regulate, then up-regulate and last down-regulate. 2hrs group hadobvious down-regulation and 6hrs group had the lowest value, and 8hrs groupshowed up-regulation, 24hrs group got the peak value, then the expression of AQP-4gradually down-regulated, 7d group it was still higher than normal;②The correlationcoefficients between expression of AQP-4 and the parameters of diffusion imagingwere low, they were 0.2902～0.4475. Conclusion: The dynamic evolution of AQP-4of the ependymal cells is first down-regulated, then up-regulated and lastdown-regulated, this trend make the fund for further pharmacology study.更多还原