Purpose

Contrast-enhanced magnetic resonance imaging (MRI) has the potential to replace angiographic evaluation of atherosclerosis. While studies have investigated contrast agent (CA) uptake in atherosclerotic plaques, exact CA spatial distribution on a microscale is elusive. The purpose of this study was to investigate the microdistribution of gadolinium (Gd)- and iron (Fe) oxide-based CA in atherosclerotic plaques of New Zealand White rabbits.

Procedures

The study was performed as a post hoc analysis of archived tissue specimens obtained in a previous in vivo MRI study conducted to investigate signal changes induced by very small superparamagnetic iron oxide nanoparticles (VSOP) and Gd-BOPTA. For analytical discrimination from endogenous Fe, VSOP were doped with europium (Eu) resulting in Eu-VSOP. Formalin-fixed arterial specimens were cut into 5-μm serial sections and analyzed by immunohistochemistry (IHC: Movat’s pentachrome, von Kossa, and Alcian blue (pH 1.0) staining, anti-smooth muscle cell actin (anti-SMA), and anti-rabbit macrophage (anti-RAM-11) immunostaining) and elemental microscopy with laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and synchrotron radiation μX-ray fluorescence (SR-μXRF) spectroscopy. Elemental distribution maps of Fe, Eu, Gd, sulfur (S), phosphorus (P), and calcium (Ca) were investigated.

Results

IHC characterized atherosclerotic plaque pathomorphology. Elemental microscopy showed S distribution to match the anatomy of arterial vessel wall layers, while P distribution corresponded well with cellular areas. LA-ICP-MS revealed Gd and Fe with a limit of detection of ~ 0.1 nmol/g and ~ 100 nmol/g, respectively. Eu-positive signal identified VSOP presence in the vessel wall and allowed the comparison of Eu-VSOP and endogenous Fe distribution in tissue sections. Extracellular matrix material correlated with Eu signal intensity, Fe concentration, and maximum Gd concentration. Eu-VSOP were confined to endothelium in early lesions but accumulated in cellular areas in advanced plaques. Gd distribution was homogeneous in healthy arteries but inhomogeneous in early and advanced plaques. SR-μXRF scans at 0.5 μm resolution revealed Gd hotspots with increased P and Ca concentrations at the intimomedial interface, and a size distribution ranging from a few micrometers to submicrometers.

Conclusions

Eu-VSOP and Gd have distinct spatial distributions in atherosclerotic plaques. While Eu-VSOP distribution is more cell-associated and might be used to monitor atherosclerotic plaque progression, Gd distribution indicates arterial calcification and might help in characterizing plaque vulnerability.

中文翻译：

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LA-ICP-MS 和 SR-μXRF 成像测定动脉粥样硬化斑块中磁共振成像造影剂的微分布

目的

对比增强磁共振成像 (MRI) 有可能取代动脉粥样硬化的血管造影评估。虽然研究已经调查了动脉粥样硬化斑块中造影剂 (CA) 的摄取，但在微观尺度上精确的 CA 空间分布是难以捉摸的。本研究的目的是研究基于氧化钆 (Gd) 和氧化铁 (Fe) 的 CA 在新西兰白兔动脉粥样硬化斑块中的微分布。

程序

该研究是对先前体内MRI 研究中获得的存档组织标本的事后分析，该研究旨在研究由非常小的超顺磁性氧化铁纳米颗粒 (VSOP) 和 Gd-BOPTA 引起的信号变化。为了与内源性 Fe 进行分析区分，VSOP 掺杂有铕 (Eu)，从而得到 Eu-VSOP 。将福尔马林固定的动脉标本切成 5 μm 连续切片，并通过免疫组织化学（IHC：Movat 五色、von Kossa 和阿尔新蓝（pH 1.0）染色、抗平滑肌细胞肌动蛋白（抗 SMA）和抗兔巨噬细胞（抗 RAM-11）免疫染色）和元素显微镜与激光烧蚀电感耦合等离子体质谱 (LA-ICP-MS) 和同步辐射 μX 射线荧光 (SR-μXRF) 光谱。研究了 Fe、Eu、Gd、硫 (S)、磷 (P) 和钙 (Ca) 的元素分布图。

结果

IHC 表征了动脉粥样硬化斑块的病理形态。元素显微镜显示 S 分布与动脉血管壁层的解剖结构相匹配，而 P 分布与细胞区域很好地对应。LA-ICP-MS 显示 Gd 和 Fe 的检测限分别为 ~ 0.1 nmol/g 和 ~ 100 nmol/g。Eu 阳性信号确定血管壁中存在 VSOP，并允许比较组织切片中的 Eu-VSOP 和内源性 Fe 分布。细胞外基质材料与 Eu 信号强度、Fe 浓度和最大 Gd 浓度相关。Eu-VSOP在早期病变中局限于内皮，但在晚期斑块的细胞区域中积累。Gd 在健康动脉中分布均匀，但在早期和晚期斑块中分布不均匀。SR-μXRF 在 0 处扫描。

结论

Eu-VSOP 和 Gd 在动脉粥样硬化斑块中具有不同的空间分布。虽然 Eu-VSOP 分布与细胞更相关，可用于监测动脉粥样硬化斑块的进展，但 Gd 分布表明动脉钙化，可能有助于表征斑块易损性。