Application of laser scanning confocal microscopy in the analysis of particle-induced pulmonary fibrosis
Application of laser scanning confocal microscopy in the analysis of particle-induced pulmonary fibrosis
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DOI：
10.1093/toxsci/51.1.126
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发表时间：
1999-09-01
影响因子：
3.8
通讯作者：
Rogers, RA
中科院分区：
文献类型：
--
作者：
Antonini, JM;Charron, TG;Rogers, RA
激光扫描共聚焦显微镜（LSCM）使我们能够同时定量肺纤维化程度并区分完整肺组织的各种病理病变。 Lucifer Yellow 已被证明是一种理想的荧光染色剂，可用于使用 LSCM 检查嵌入肺组织的结缔组织基质成分。我们评估了 LSCM 在定量肺纤维化中的应用，并将该程序与通过测量羟脯氨酸（胶原蛋白的生化测定）来评估纤维化的更传统方法进行了比较。 CD/VAF 大鼠气管内给予二氧化硅（高度纤维化）、Fe2O3（非纤维化）和盐水（媒介物对照），剂量为 10 毫克/100 克体重。滴注后 60 天，将左肺溶解在 6 M HCl 中并测定羟脯氨酸。与 Fe2O3 组和对照组相比，二氧化硅诱导羟脯氨酸含量分别增加 58% 和 94%。将右肺叶固定，切成块，脱水，用荧光黄（0.1 mg/ml）染色，并嵌入Spurr塑料中。使用LSCM和ImageSpace软件，测量了三组中每组十个组织块的十个随机扫描的组织面积，并生成了肺部随机区域的三维重建。与 Fe2O3 组和对照组相比，二氧化硅组荧光黄染色的肺部区域分别增加了 57% 和 60%。羟脯氨酸与肺组织面积的回归分析显示显着正相关（p < 0.05），相关系数为 0.91。右肺组织的组织学分析显示，二氧化硅组有明显程度的肉芽肿性间质性肺炎，而 Fe2O3 组和对照组则没有这种情况。 Fe2O3 组和对照组之间的羟脯氨酸含量和测量的组织面积没有显着差异 (p < 0.05)。 LSCM 及其相关的先进图像分析和三维功能是一种快速定量和检查纤维化肺病且无需对组织样本进行物理破坏的替代方法。
Laser scanning confocal microscopy (LSCM) allows us to simultaneously quantitate the degree of lung fibrosis and distinguish various pathological lesions of intact lung tissue. Lucifer Yellow has been shown an ideal fluorescent stain to examine the connective tissue matrix components of embedded lung tissue with LSCM. We evaluated the use of LSCM in quantitating lung fibrosis and compared this procedure with the more traditional method of assessing fibrosis by measuring hydroxyproline, a biochemical assay of collagen. CD/VAF rats were intratracheally dosed with silica (highly fibrogenic), Fe2O3 (non-fibrogenic), and saline (vehicle control) at a high dose of 10-mg/100 g body weight. At 60 days post-instillation, the left lung was dissolved in 6 M HCI and assayed for hydroxyproline. Silica induced increases of 58% and 94% in hydroxyproline content over the Fe2O3 and control groups, respectively. The right lung lobes were fixed, sectioned into blocks, dehydrated, stained with Lucifer Yellow (0.1 mg/ml), and embedded in Spurr plastic. Using LSCM and ImageSpace software, the tissue areas of ten random scans from ten blocks of tissue for each of the three groups were measured, and three-dimensional reconstructions of random areas of lung were generated. The silica group showed increases of 57% and 60% in the lung areas stained by Lucifer Yellow over the Fe2O3 and control groups, respectively. Regression analysis of hydroxyproline vs. lung tissue area demonstrated a significant positive correlation (p < 0.05) with a correlation coefficient of 0.91. Histological analysis of right lung tissue revealed a marked degree of granulomatous interstitial pneumonitis for the silica group, which was absent in the Fe2O3 and control groups. No significant differences (p < 0.05) in hydroxyproline content and measured tissue area were observed between the Fe2O3 and control groups. LSCM, and its associated advanced image analysis and three-dimensional capabilities, is an alternative method to both quickly quantitate and examine fibrotic lung disease without physical disruption of the tissue specimen.