斑马鱼急性酒精性肝损伤的组织学分析
Summary
该方案描述了用2％乙醇处理24小时的斑马鱼幼虫肝脏的组织学分析。这种急性乙醇治疗导致肝血管的肝脂肪变性和肿胀。
Abstract
酒精性肝病（ALD）是指由于急性或慢性酒精滥用而导致的肝损害。这是酒精相关发病率和死亡率的主要原因之一，影响了美国200多万人。更好地了解酒精诱导的肝损伤的细胞和分子机制对于开发ALD的有效治疗至关重要。斑马鱼幼虫在接触2％乙醇仅24小时后出现肝脂肪变性和纤维发生，使其可用于急性酒精性肝损伤的研究。这项工作描述了斑马鱼幼虫急性乙醇治疗的程序，并表明它引起肝血管的脂肪变性和肿胀。还描述了针对斑马鱼幼虫肝组织学分析优化的苏木精和曙红（H＆E）染色的详细方案。 H＆E染色比免疫荧光具有几个独特的优点，因为它标志着所有的生物er细胞和细胞外组分同时并且可以容易地检测肝损伤，例如脂肪变性和纤维化。鉴于斑马鱼在建模毒素和病毒诱导的肝损伤以及遗传性肝脏疾病方面的用量越来越多，该协议作为所有这些研究中进行的组织学分析的参考。
Introduction
酒精饮酒引起的酒精性肝病（ALD）是酒精相关发病率和死亡率的主要原因。在美国，近一半的肝脏疾病死亡涉及酒精1 ，ALD负责近1/3的肝脏移植2 。 ALD有广谱。脂肪变性，其特征在于肝细胞中过量的脂质积累，发生在重度饮酒的早期阶段，并且在酒精停止使用时是可逆的。在遗传和环境因素和持续饮酒的影响下，肝脂肪变性可能发展为酒精性肝炎，最终导致肝硬化3 。使用啮齿动物ALD模型的研究为疾病提供了深刻的见解，但它们有局限性（参考文献3 ）。口服酒精饮食只会引起啮齿动物脂肪变性4/ sup> 5 。炎症和纤维化的发展需要第二次侮辱6,7或慢性胃内输注，这是侵入性和技术上具有挑战性的8,9 。硬糖斑马鱼也发生肝损伤，对慢性和急性酒精治疗10,11,12,13,14,15。特别是，幼虫斑马鱼是一种有吸引力的互补模式生物，其中研究急性酒精性肝损伤10,11,13,15。斑马鱼肝功能并在4天内产生乙醇代谢的关键酶（dpf）13,16,17。乙醇可以直接加入到水中，暴露于2％乙醇24小时足以在斑马鱼幼虫13,15中诱导肝脂肪变性和纤维化反应。
据报道，用2％乙醇处理24小时导致斑马鱼幼虫13的组织乙醇浓度为80mM。其他人已经表明幼虫耐受这一浓度，并且在处理的动物中看到的肝脏表型对乙醇暴露具有特异性11,13,15,18。然而，由于80mM在人体中几乎是致命的19 ，重要的是评估乙醇处理的斑马鱼的肝脏组织学和dete对人类的生理相关性。
斑马鱼幼虫的快速外部发育和半透明使得有可能实时和固定样品中肝脏中酒精的作用。细胞型特异性荧光转基因品系的可获得性以及共聚焦显微镜近期的进展有助于研究不同肝细胞类型如何改变其响应于急性乙醇处理的形态和行为11,15。然而，当研究肝组织学时，荧光转基因斑马鱼的共聚焦成像不能完全替代苏木精和曙红（H＆E）染色。使用转基因斑马鱼同时标记所有肝细胞类型需要产生个体转基因品系，每个转基因品系用独特的荧光团标记一个肝细胞类型。将不同的转基因背景引入同一条鱼需要繁殖多代，这是耗时且昂贵的。需要额外的免疫荧光染色来检测细胞外基质成分。另一方面，H＆E染色同时标记所有肝细胞类型和细胞外基质组分，从而提供肝脏的概况20 。此外，它容易显示肝脏疾病的几种组织病理学特征，如肝细胞死亡，脂肪变性和纤维化。虽然H＆E是哺乳动物肝脏组织学中的常规染色体，但它并不常用于斑马鱼肝脏研究，而且协议不太成熟。
这项工作描述了斑马鱼幼虫急性乙醇治疗方案和H＆E染色后续组织学分析。 H＆E染色方案可用于所有肝脏发育和功能研究。此外，石蜡切片可用于免疫组织化学和其他特殊治疗包括肝脏病理学，包括三色染色，网状蛋白染色等 。
Protocol
AB WT成年和幼虫斑马鱼根据“实验动物护理和使用指南”（美国国家卫生研究院出版号86-23，1985年修订版）保持在标准条件21 。其使用由辛辛那提儿童医院医疗中心（CCHMC）的机构动物护理和使用委员会批准。 1.准备解决方案 准备蛋水。 通过将40g商业海盐溶解在1L双蒸水（ddH 2 O）中来制备储备盐溶液。搅拌至所有盐?…
Representative Results
10％缓冲福尔马林和4％多聚甲醛（PFA）是用于组织学实践的两种最常见的固定剂。然而，他们没有为斑马鱼肝组织提供最佳的固定结果（ 图1和表1 ）。用10％福尔马林或4％PFA固定经常导致收缩，在肝脏和周围组织之间产生大的间隙（ 图1A ， B ; 图1B提供组织收缩的实例）。肝组?…
Discussion
目前的方案描述了斑马鱼幼虫急性乙醇处理的详细程序和随后的H＆E染色组织病理学分析。急性乙醇治疗应不迟于受精后96小时进行，因为这是斑马鱼肝开始表达酒精代谢酶13的阶段 。 2％乙醇是幼虫可耐受的最大剂量13,14 。乙醇处理的幼虫在处理8小时后开始出现肝脂肪变性，发生脂肪变性的幼虫的百分比持续上升，直到24小时连续处理14 。斑?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者想在斑马鱼国际资源中心承认Katy Murray博士; CCHMC的Stacey Huppert博士和Kari Huppert博士就协议提供了有用的建议;和CCHMC兽医服务，用于养鱼。这项工作得到NIH授权R00AA020514和CCHMC（CY）儿科基因组学中心的研究资助。它也得到了NIH授予P30 DK078392（综合形态核心）消耗性疾病研究核心中心在辛辛那提的支持。
Materials
| 1.5 mL centrifuge tubes | E & K Scientific | 280150 | |
| 15 mL conical tubes | VWR International | 89039-664 | |
| 50 mL conical tubes | VWR International | 89039-658 | |
| 95% ethanol | Decon Labs, Inc. | 2801 | Flammable |
| Acetic acid | Newcomer Supply | 10010A | Irritant |
| Agarose | Research Products International | 9012-36-6 | |
| Aluminum jar rack holder | Newcomer Supply | 5300JRK | |
| Bacteriological petri dishes with lid | Corning | 351029 | |
| Biopsy pads | Simport | M476.1 | |
| Charged slides | Fisher Scientific | 12-550-16 | |
| Clear mounting media | Fisher Scientific | 8310-16 | Can be substituted with other clear mounting media |
| Commercial sea salts | Instant Ocean | SS15-10 | |
| Disposable microtome blades | Fisher Scientific | 4280L | |
| Dissecting microscope | Leica Biosystems | Leica Mz 95 | |
| Enclosed tissue processor | Leica Biosystems | ASP300 S | |
| Eosin-Phloxine stain set | Newcomer Supply | 1082A | |
| Ethyl alcohol | Sigma-Aldrich | E7023 | Flammable |
| Formaldehyde solution, ACS reagent, 37 WT. % in H20, contains 10-15% methanol as stabilizer (to prevent polymerization) | Sigma-Aldrich | 252549 | A suspected carcinogen; irritant |
| Formalin, Buffered, 10% | Fisher Scientific | SF100-4 | A suspected carcinogen; irritant |
| Graduated media bottle | VWR International | 16159-520 | |
| Harris hematoxylin | Poly Scientific R&D Corp. | s212 | Irritant |
| Histology molds | Sakura Finetek USA Inc | 4557 | |
| Hot plate/Stirrer | VWR International | 47751-148 | |
| Hydrochloric acid | Fisher Scientific | A144 | Irritant |
| Incubator | VWR International | 97058-220 | |
| Insulin syringes | BD Medical | BD-309301 | |
| Inverted compound microscope | Carl Zeiss Microscopy | 491912-9850-000 | |
| Isopropanol | Newcomer Supply | 12094E | Flammable |
| Methylene blue | Sigma-Aldrich | M9140 | Irritant |
| Microtome | Leica Biosystems | Leica Jung BioCut 2035 | |
| Nutating mixer | VWR International | 82007-202 | |
| Paraformaldehyde | Sigma-Aldrich | P6148-1KG | A suspected carcinogen; irritant |
| Pasteur pipet | VWR International | 53283-916 | |
| Pipette pump (10 mL) | VWR International | 53502-233 | |
| Potassium chloride (KCl) | Sigma-Aldrich | P9541 | |
| Potassium phosphate, monobasic (KH2PO4) | Sigma-Aldrich | P9791 | |
| Razor blades | Grainger | 4A807 | |
| Slide Staining Kit | Newcomer Supply | 5300KIT | |
| Sodium chloride (NaCl) | Sigma-Aldrich | S3014 | |
| Sodium hydroxide (NaOH) | Fisher BioReagents | S318-500 | Very hazardous |
| Sodium phosphate, dibasic (Na2HPO4) | Sigma-Aldrich | S3264 | |
| Stainless steel strainer (5 inch diameter) | Adaptive Science Tools | L0906045in | |
| Tissue cassettes | Simport | M505.12 | |
| Tissue embedding center | Sakura Finetek USA Inc | #5100 | |
| Tissue wipers, 1-Ply | Fisher Scientific | 06666A | |
| Transfer pipets | Fisher Scientific | 137117M | |
| Tricaine powder/Ethyl 3-aminobenzoate methanesulfonate salt | Sigma-Aldrich | A5040 | Irritant |
| Tris base, primary standard and buffer | Sigma-Aldrich | T1503 | |
| Wash bottle, low-density polyethylene, wide mouth | Nalge Nunc International | 2402-0750 | |
| Xylenes | Fisher Scientific | X3S-4 | Irritant |
Riferimenti
- Yoon, Y. H., Chen, C. M., Yi, H. Y. . Surveillance report #100: Liver cirrhosis mortality in the United States: National, State, and regional trends. , 2000-2011 (2014).
- Singal, A. K., et al. Evolving frequency and outcomes of liver transplantation based on etiology of liver disease. Transplantation. 95 (5), 755-760 (2013).
- Louvet, A., Mathurin, P. Alcoholic liver disease: mechanisms of injury and targeted treatment. Nat Rev Gastroenterol Hepatol. 12 (4), 231-242 (2015).
- Ki, S. H., et al. Interleukin-22 treatment ameliorates alcoholic liver injury in a murine model of chronic-binge ethanol feeding: role of signal transducer and activator of transcription 3. Hepatology. 52 (4), 1291-1300 (2010).
- Tsuchiya, M., et al. Interstrain differences in liver injury and one-carbon metabolism in alcohol-fed mice. Hepatology. 56 (1), 130-139 (2012).
- Koteish, A., Yang, S., Lin, H., Huang, X., Diehl, A. M. Chronic ethanol exposure potentiates lipopolysaccharide liver injury despite inhibiting Jun N-terminal kinase and caspase 3 activation. J Biol Chem. 277 (15), 13037-13044 (2002).
- Leo, M. A., Lieber, C. S. Hepatic fibrosis after long-term administration of ethanol and moderate vitamin A supplementation in the rat. Hepatology. 3 (1), 1-11 (1983).
- Tsukamoto, H., et al. Severe and progressive steatosis and focal necrosis in rat liver induced by continuous intragastric infusion of ethanol and low fat diet. Hepatology. 5 (2), 224-232 (1985).
- Tsukamoto, H., Mkrtchyan, H., Dynnyk, A. Intragastric ethanol infusion model in rodents. Methods Mol Biol. 447, 33-48 (2008).
- Howarth, D. L., Passeri, M., Sadler, K. C. Drinks like a fish: using zebrafish to understand alcoholic liver disease. Alcohol Clin Exp Res. 35 (5), 826-829 (2011).
- Howarth, D. L., Yin, C., Yeh, K., Sadler, K. C. Defining hepatic dysfunction parameters in two models of fatty liver disease in zebrafish larvae. Zebrafish. 10 (2), 199-210 (2013).
- Lin, J. N., et al. Development of an Animal Model for Alcoholic Liver Disease in Zebrafish. Zebrafish. , (2015).
- Passeri, M. J., Cinaroglu, A., Gao, C., Sadler, K. C. Hepatic steatosis in response to acute alcohol exposure in zebrafish requires sterol regulatory element binding protein activation. Hepatology. 49 (2), 443-452 (2009).
- Tsedensodnom, O., Vacaru, A. M., Howarth, D. L., Yin, C., Sadler, K. C. Ethanol metabolism and oxidative stress are required for unfolded protein response activation and steatosis in zebrafish with alcoholic liver disease. Dis Model Mech. 6 (5), 1213-1226 (2013).
- Yin, C., Evason, K. J., Maher, J. J., Stainier, D. Y. The bHLH transcription factor Hand2 marks hepatic stellate cells in zebrafish: Analysis of stellate cell entry into the developing liver. Hepatology. , (2012).
- Lassen, N., et al. Molecular cloning, baculovirus expression, and tissue distribution of the zebrafish aldehyde dehydrogenase 2. Drug Metab Dispos. 33 (5), 649-656 (2005).
- Reimers, M. J., Hahn, M. E., Tanguay, R. L. Two zebrafish alcohol dehydrogenases share common ancestry with mammalian class I, II, IV, and V alcohol dehydrogenase genes but have distinct functional characteristics. J Biol Chem. 279 (37), 38303-38312 (2004).
- Zhang, C., Ellis, J. L., Yin, C. Inhibition of vascular endothelial growth factor signaling facilitates liver repair from acute ethanol-induced injury in zebrafish. Dis Model Mech. , (2016).
- Vonghia, L., et al. Acute alcohol intoxication. Eur J Intern Med. 19 (8), 561-567 (2008).
- Wittekind, D. Traditional staining for routine diagnostic pathology including the role of tannic acid. 1. Value and limitations of the hematoxylin-eosin stain. Biotech Histochem. 78 (5), 261-270 (2003).
- Westerfield, M. . The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio). , (2007).
- Theise, N. D. Histopathology of alcoholic liver disease. Clinical Liver Disease. 2 (2), (2013).
- Lorent, K., et al. Inhibition of Jagged-mediated Notch signaling disrupts zebrafish biliary development and generates multi-organ defects compatible with an Alagille syndrome phenocopy. Development. 131 (22), 5753-5766 (2004).
- Huang, M., Xu, J., Shin, C. H. Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration. J Vis Exp. (111), (2016).
- Paredes, J. F., Lopez-Olmeda, J. F., Martinez, F. J., Sanchez-Vazquez, F. J. Daily rhythms of lipid metabolic gene expression in zebra fish liver: Response to light/dark and feeding cycles. Chronobiol Int. 32 (10), 1438-1448 (2015).
- Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. Method for isolation of PCR-ready genomic DNA from zebrafish tissues. Biotechniques. 43 (5), 610-614 (2007).
- van der Velden, Y. U., et al. The serine-threonine kinase LKB1 is essential for survival under energetic stress in zebrafish. Proc Natl Acad Sci U S A. 108 (11), 4358-4363 (2011).
Citazione di questo articolo
Ellis, J. L., Yin, C. Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish. J. Vis. Exp. (123), e55630, doi:10.3791/55630 (2017).