一个标准化的方法肝窦内皮细胞的分析及其窗孔用扫描电子显微镜
Summary
The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.
Abstract
Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures – both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.
Introduction
肝窦内皮细胞（LSECs）是高度分化的内皮细胞排列在肝窦的壁。 LSECs均穿有穿孔是非diaphragmed，跨细胞孔径为50-250纳米。高达LSECs的表面的20％被覆盖穿孔，这通常是在几十组到几百称为筛板1-3（ 图1）。开窗使血浆和血液和肝细胞之间nanosubstrates的转移，创造一个高效的超滤系统。窗孔是动态的结构 – 无论它们的尺寸和/或数量可以响应于各种生理状态，药物和疾病而改变。例如，穿孔较大在禁食比在进食状态4; 2-二- iodoamphetamine增加开窗号; 5,6-和尺寸的减小和穿孔的数目每细胞发生衰老和许多疾病状态7-13 </sup>。窗孔的尺寸和数量的精确测量是理解如何LSEC形态是由各种疾病，中毒性和生理状态的影响重要;它们对肝功能的影响;和用于显影开窗调制治疗干预1。
窗孔的研究是困难的。窗孔的直径在于传统光学显微镜的分辨率下面，所以无论是在完整的肝组织或培养LSECs以前只观察用电子显微镜已经成为可能。扫描电子显微镜（SEM）已最经常被用于研究开窗尺寸，频率和孔隙率（LSEC膜是受窗孔穿孔的百分比），因为SEM允许观测大面积的数千内皮表面和测量，如果不是几万窗孔的。尽管它的效用，这是从报告的结果的SEM为基础的研究为LSEC参数如开窗大小，数量，频率和孔隙率有很大的不同在文献中（ 表1）。
开窗和筛板是脆弱的结构，其合同，打破，扩张或标本制备过程中凝聚，从而精心加工是需要保持其完整性。升高灌注压14;固定液和缓冲区15的不正确的渗透压;固定不充分或固定时间;和后固定脱水和干燥的速度是处理用于SEM的所有领域，可能产生伪影，与保存超微结构的干扰（ 图2）。窗孔的损失（'抛出窗外'）和开窗收缩可发生定影不良的结果，从而降低开窗直径和孔的孔隙率。方法，提高试样的保存用于SEM分析先前已经描述15-17和将要讨论的这里，关于如何提高标本保存额外的提示。检体保存的主要目标是从血窦除去血液，使得LSEC的表面可以可视化，并避免从任一高压或延迟固定的LSEC损坏。固定剂经门静脉全肝灌注是肝脏固定的首选方法。如在详细描述别处16,18灌注必须在低压力下进行（例如，10厘米H 2 O），以避免压力相关灌注艺术品和损坏LSEC，典型表现内于细胞膜一样大的差距。然而，合理的固定通常可以使用从人类和动物的肝活检的针灌注获得，如在别处19进行详细描述。该技术包括直接注射入固定液的组织，直到血冲出样品和组织是坚定的和固定的。样品电子显微镜的固定需要被法律约束ormed尽快以下血流停止，以防止发生由于肝脏极其迅速自溶过程的结果超微结构改变。
我们还提出，最大限度地减少包含伪影，和标准化窗孔的测量图像分析的方法。变化血窦为显微照片的选择，人工制品的图像分析，并且小区区域的测量孔隙度和开窗频率已导致在已发表的结果重大差异。评估和窗孔和数据呈现的最低要求的测量标准化的做法没有得到明确解决在文献中以前4,10,20-31。
Protocol
Representative Results
Discussion
精确地和可重复地测量所述肝窦内皮的状态的能力是在理解这些高度特化的细胞的生物学的一个重要步骤。如结构照明显微镜32，原子力显微镜33和d- STORM（直接随机光学Reconstrucion显微镜）34更新的技术将赋予重要的信息对这些细胞在体外的形态，但SEM仍然是主要的方法来可视化和在测量它们的结构原位 。
最关键和技术挑战性的一?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors have no acknowledgements.
Materials
| Name of material | Company | Catalogue Number | Comments |
| EM grade Glutaraldehyde | ProSciTech | C001 | Store stock at -20 C until needed, avoid refreeze |
| Paraformaldehyde powder | Sigma Aldrich | 158127 | Always prepare Paraformaldehyde fresh |
| Sodium Cacodylate powder | Sigma Aldrich | C0250 | Prepare 0.2 M stock, pH 7.4 by dissolving powder in dH2O, used mostly at 0.1 M by preparing 1:2 dilution |
| Calcium Chloride | Sigma Aldrich | C1016 | Prepare 1 M CaCl2by dissolving powder in dH2O |
| Osmium tretroxide | ProSciTech | C011 | Wash ampoules in weak acid prior to use to avoid contamination. Prepare 2 % stock in glass bottle |
| Ethanol- Absolute | Sigma Aldrich | 459836 | 100 % Ethanol must be high grade and stored with Molecular Sieve |
| Other grades of Ethanol | Labtech | EL5 | Prepare graded Ethanols with dH2O |
| Hexamethyldisilazane | Sigma Aldrich | 52619 | Allow to reach room temperature before use |
| Cannulas | Terumo | TSROX1832C, TSROX2225C, TSROX2419C | 18 G is suitable for most rats, 22 G is suitable for most mice, but it is good to have a few 24 G on hand in case of very small mice |
| Conductive Carbon tape | ProSciTech | IA0201 | |
| Carbon Paint | ProSciTech | I003 | |
| Ketamine | Must be optained under licence | ||
| Xylazine | Must be obtained under licence | ||
| Molecular Sieve | Sigma Aldrich | 208647 | Removes water from the 100 % Ethanol |
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