Calvo W J, Lieber B B, Hopkins L N, Wakhloo A K
Toshiba Stroke Research Center, NY, USA.
AJNR Am J Neuroradiol. 2001 Apr;22(4):691-7.
Standard tissue staining using the lipid dye Oil-Red-O has been previously applied to stain vessel specimens, which were embolized with a mixture of n-butyl 2-cyanoacrylate (NBCA) and oil (Lipiodol). That technique, however, results in nonspecific and nonquantitative staining that does not provide the necessary differentiation between NBCA and Lipiodol. We present an innovative staining procedure that quantifies NBCA within treated tissues.
An arteriovenous malformation (AVM) model in swine was used to evaluate the polymerization characteristics of various ratios of Lipiodol/NBCA/glacial acetic acid (GAA) mixtures. To determine the depth of NBCA penetration within the AVM model and to characterize the polymerization patterns of various mixtures within the vessel, histologic cross- and longitudinal sections were prepared for microscopy. These paraffin-embedded tissue sections were stained with a europium aryl-beta-diketone complex (TEC) to improve differentiation between NBCA and Lipiodol. Quantification of NBCA and Lipiodol within the lumen of rete cross-sections was accomplished using image analysis software to determine percent luminal area occluded by embolization.
Upon application of TEC, intense europium fluorescence was seen when the tissue samples were excited by low-power UV light (excitation at 365 nm; emission at 614 nm). The area of europium intensity within the lumen corresponded to NBCA concentration, and addition of GAA aided the NBCA distribution throughout the lumen without affecting fluorescence intensity. It was seen that NBCA could be easily differentiated from Lipiodol and that quantification could be readily performed on these sections because of the improved differentiation. For the case of a 50:50 (vol. %) mixture with an added 20 microL of GAA, luminal area distribution of Lipiodol, NBCA, and blood products was 42.6 +/- 3.5%, 33.8 +/- 5.7%, and 23.7 +/-2.7%, respectively.
The rare earth metal europium, when added as a fluorescent chelate compound to histologic tissue sections, allowed for differentiation between NBCA and Lipiodol with good detail. These results have facilitated further characterization of NBCA polymerization for the use of AVM embolization.
此前已应用脂质染料油红O进行标准组织染色，以对用氰基丙烯酸正丁酯（NBCA）与油（碘油）混合物栓塞的血管标本进行染色。然而，该技术会导致非特异性和非定量染色，无法在NBCA和碘油之间实现必要的区分。我们提出了一种创新的染色方法，可对处理后的组织中的NBCA进行定量。
使用猪的动静脉畸形（AVM）模型来评估碘油/NBCA/冰醋酸（GAA）不同比例混合物的聚合特性。为确定NBCA在AVM模型内的渗透深度，并表征血管内不同混合物的聚合模式，制备了组织学横断面和纵断面切片用于显微镜检查。这些石蜡包埋的组织切片用铕芳基β - 二酮配合物（TEC）染色，以改善NBCA和碘油之间的区分。使用图像分析软件对网状横断面管腔内的NBCA和碘油进行定量，以确定栓塞导致的管腔面积闭塞百分比。
应用TEC后，当组织样本用低功率紫外光激发（激发波长365nm；发射波长614nm）时，可见强烈的铕荧光。管腔内铕强度区域对应于NBCA浓度，添加GAA有助于NBCA在整个管腔内分布，而不影响荧光强度。可以看到，NBCA能够很容易地与碘油区分开来，并且由于区分得到改善，这些切片上的定量操作也能够很容易地进行。对于添加20μL GAA的50:50（体积百分比）混合物，碘油、NBCA和血液产物的管腔面积分布分别为42.6±3.5%、33.8±5.7%和23.7±2.7%。
当作为荧光螯合物添加到组织学组织切片中时，稀土金属铕能够很好地区分NBCA和碘油。这些结果有助于进一步表征用于AVM栓塞的NBCA聚合情况。
