Influence of fixation on CA4+ contrast enhanced microCT of articular cartilage and subsequent feasibility for histological evaluation.
Influence of fixation on CA4+ contrast enhanced microCT of articular cartilage and subsequent feasibility for histological evaluation.
复制标题
DOI：
--
复制
发表时间：
2021
影响因子：
2.2
通讯作者：
Xueqin Gao;Amit N. Patwa;Zhenhan Deng;Hajime Utsunomiya;M. Grinstaff;Joseph J. Ruzbarsky;B. Snyder;S. Ravuri;M. Philippon;J. Huard
中科院分区：
文献类型：
--
作者：
Xueqin Gao;Amit N. Patwa;Zhenhan Deng;Hajime Utsunomiya;M. Grinstaff;Joseph J. Ruzbarsky;B. Snyder;S. Ravuri;M. Philippon;J. Huard
CA4+ 是一种新型阳离子碘造影剂，用于增强显微 CT (CECT)。在本研究中，我们比较了 CA4+ CECT 对未固定和中性缓冲福尔马林 (NBF) 固定的兔股骨远端软骨在造影剂扩散 8、24 和 30 小时后的软骨定量。还通过CECT研究了PBS浸泡和脱钙后CA4+与软骨结合的稳定性。我们进一步评估了 CA4+ CECT 后软骨组织学和免疫组织化学的可行性。对比增强的 CA4+ 标记的未固定和 NBF 固定的软骨组织有利于关节软骨定量和准确的形态学评估。 NBF 固定组织比未固定组织表现出更高的软骨强度和与软骨下骨不同的成像特征，同时即使在用 10% EDTA 脱钙后也能保持稳定的结合。经过 CECT 成像和脱钙后，用 CA4+ 标记的未固定组织可进行 H&E、阿尔新蓝和番红 O 染色以及 Col2 免疫组织化学染色。相比之下，CA4+ 标记的 NBF 固定软骨只能进行 H&E 和阿新蓝染色，并且 CA4+ 标记会干扰下游免疫组织化学和番红 O 染色，这可能是由于其带正电荷。总之，NBF 固定组织的 CA4+ CECT 提供了高质量的 microCT 软骨图像，并允许方便的定量以及脱钙后可行的下游 H&E 和阿尔新蓝染色。未固定组织的 CA4+ CECT 使研究人员能够以成本、时间和劳动力高效的方式获得一组动物的定量 microCT 以及软骨组织学和免疫组织化学数据。
CA4+ is a novel cationic iodinated contrast agent utilized for contrast-enhanced microCT (CECT). In this study, we compared CA4+ CECT for cartilage quantification of unfixed and neutral buffered formalin (NBF)-fixed rabbit distal femur cartilage after 8-, 24- and 30-hours of contrast agent diffusion. The stability of CA4+ binding to cartilage after PBS soak and decalcification was also investigated by CECT. We further assessed the feasibility of cartilage histology and immunohistochemistry after CA4+ CECT. Contrast-enhanced CA4+ labeled unfixed and NBF-fixed cartilage tissues facilitate articular cartilage quantification and accurate morphological assessment. The NBF fixed tissues demonstrate higher cartilage intensity and imaging characteristics distinct from subchondral bone than unfixed tissues while maintaining stable binding even after decalcification with 10% EDTA. The unfixed tissues labeled with CA4+, after CECT imaging and decalcification, are amenable to H&E, Alcian blue, and Safranin O staining, as well as Col2 immunohistochemistry. In contrast, only H&E and Alcian blue staining can be accomplished with CA4+ labeled NBF fixed cartilage, and CA4+ labeling interferes with downstream immunohistochemistry and Safranin O staining, likely due to its positive charge. In conclusion, CA4+ CECT of NBF fixed tissues provides high quality microCT cartilage images and allows for convenient quantification along with feasible downstream H&E and Alcian blue staining after decalcification. CA4+ CECT of unfixed tissues enables researchers to obtain both quantitative microCT as well as cartilage histology and immunohistochemistry data from one set of animals in a cost-, time-, and labor-efficient manner.