Name: repeated blood collection for blood tests in adult zebrafish
Uploaded: 2015-08-30T14:00:00.000+00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Repeated Blood Collection for Blood Tests in Adult Zebrafish at JoVE.com

This work was supported by JSPS KAKENHI Grant Number 25860294 and 25590073. We would like to thank Ms. Yui Namie for the hand-drawn illustration, and Mr. Koshi Kataoka and Ms. Sayuri Ichikawa for assistance with the zebrafish maintenance.

所有动物的程序是由三重大学伦理委员会批准，并与国际准则根据日本动物福利法规"法对福利和动物管理"（日本国环境）进行，并遵守。 1.准备了针注:在麻醉状态下进行所有的实验，并尽一切努力，尽量减少痛苦。安乐死，鱼浸入冰 - 水浴（5份冰/ 1份水在≤4℃）≥20分钟。 <ol><li>通过抽1.0毫米外直径的玻璃毛细管用针拔出器（图1A）制备玻璃微毛细管针。 </li><li>切斜用细剪刀（ 图1B）针的提示。提供了理想尖端直径应该大约100 - 200微米（图1C）。如果尖地径r是太窄，血液不会进入针。 </li><li>溶解肝素生理盐水至5毫克/毫升的浓度。 </li><li>放置一个预切针在吸气管组件的鼻甲端和保持吸嘴在口中，或将针连接到灯泡分配器。浸入针尖到肝素溶液，并通过抽吸并通过溶液（图1D和1E）吹heparinize针头。 注意:吸气管组件已经用于斑马鱼精子冷冻保存16。长的橡胶管可以阻止任何血液飞入嘴。设置一个过滤器在所述管的中途可避免危险。 </li><li>存储在一个10厘米的培养皿中，然后风干的肝素化的针至少1小时。甲大量的针可以提前准备。 </li></ol> 2.麻醉<ol><li>通过将200个ml的鱼水与100微升2-苯氧乙醇制备的塑料小情况下，麻醉剂溶液（2-PE）。麻醉剂的终浓度为500ppm。 注意！ 2-PE具有快速的效果。 </li><li>除去鱼（AB株）从循环系统期望数量。 </li><li>用网鱼转移到麻醉剂1 - 2分钟（ 图1F）。观察鱼慢慢游，水平传播的胸鳍，一声惊呼，具有快速的鳃盖运动在1分钟内。 </li><li>随着时间的推移，观察鱼躺在外壳的底部，并最终停止游泳。当鱼停下来喘气和鳃盖动作缓慢麻醉的手术平面达到。在这一点上，鱼准备采血。 </li><li>使用撇渣器从2-PE抬起麻醉鱼并轻轻将其放置在纸巾浸渍于该麻醉药（图1G）。盖鱼的头部软组织文中还浸泡2-PE的解决方案，以防止眼睛干涩和使用另一种柔软的干纸巾轻轻擦干体表。 </li></ol> 3.采血<ol><li>发生在吸气管组件（或灯泡分配器）的管口端的肝素针和保持吸气管组件在嘴里的喉舌结束。 </li><li>把握鼻甲端和针一起，并小心地用针尖移除干扰鳞。将针头在30 - 45°角插入采血部位。避免胃肠道（ 图1H）穿刺。在使用灯泡分配器的，按用拇指和中指的灯泡，并阻断孔在与第一手指的灯泡的端部，再插入针如上​​所述。 注意:站点采血是沿着主体轴线和后向中的背主动脉的区域中的肛门。背主动脉（DA）和所述后部主静脉（PCV）只是腹侧脊柱（ 图2）。 </li><li>开始吸衔嘴吸引管组件的端部时的针毡接触脊柱。如果血液不涨，用手移动针尖巧妙地鼓励血流。需要注意的是，一旦血液上升到针，立即停止摇动吸轻轻地（ 图1I）。在使用灯泡分配器的，释放灯泡的压力以吸入血液。 </li><li>观察血液会慢慢上升到针以脉动方式不经抽吸，这是动脉血压，因为可能的。因此，就没有必要吸如果针正确地穿透动脉。 </li><li>停止抽吸采集的血液相应的卷后。从鱼取出针，用柔软的纸巾停止任何出血按下穿刺部位。 20秒的指压（图1J） -在大约10观察出血停止。 </li><li>后出血停止后，立即将鱼放回转移到CLE在温水（〜28℃）坦克。帮助鱼儿轻轻纷飞水对鱼鳃，直到它开始游泳恢复。 注意:保持高达5 postsampling鱼在2升罐和罐连接到循环系统充氧的鱼。除抗生素鱼水是没有必要的。保持正常的住房和喂鱼。 </li><li>排出血液从针放在一张封口膜（ 图1K）的洁净区。 </li><li>使用任何商业手持式血糖仪（ 图1L）测量血糖。 注意:血糖仪使用葡萄糖脱氢酶黄素腺嘌呤二核苷酸电极，并且需要的0.6微升的样品体积。 <ol><li>插入试纸完全进入计和直接接触血滴。自动观察血液抽取到测试条，将获得的血糖结果5秒上的显示区域。记录结果并丢弃测试条。用一个新的测试条为每个测量。 </li></ol></li><li> （可选步骤）采取准确量的血液由吸液和血液转移到微离心管，用于进一步的分析（血红蛋白，甘油三酯（TG），总胆固醇等 ）。如果需要的话，稀释的全血从一个钓鱼用盐水。离心血液样品3分钟，在680×g离心在RT和收获血浆。转移等离子体进新管中。在这一点上，它准备在生化分析中使用。 </li></ol>

<ol>
	<li>Lieschke, G., Currie, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat Rev Genet. 8 (5), 353-367 (2007).</li><li>Penberthy, W. T., Shafizadeh, E., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+human+disease.">The zebrafish as a model for human disease.</a> Front Biosci. 7, d1439-d1453 (2002).</li><li>Stachura, D. L., Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+dissection+of+zebrafish+hematopoiesis.">Cellular dissection of zebrafish hematopoiesis.</a> Methods Cell Biol. 101, 75-110 (2011).</li><li>Jagadeeswaran, P., Sheehan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+blood+coagulation+in+the+zebrafish.">Analysis of blood coagulation in the zebrafish.</a> Blood Cells Mol Dis. 25 (3-4), 239-249 (1999).</li><li>Jagadeeswaran, P., Sheehan, J. P., Craig, F. E., Troyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+zebrafish+thrombocytes.">Identification and characterization of zebrafish thrombocytes.</a> Br J Haematol. 107 (4), 731-738 (1999).</li><li>Lieschke, G. J., Oates, A. C., Crowhurst, M. O., Ward, A. C., Layton, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+functional+characterization+of+granulocytes+and+macrophages+in+embryonic+and+adult.">Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult.</a> Blood. 98 (10), 3087-3096 (2001).</li><li>Iwanami, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+understanding+the+evolution+of+the+vertebrate+immune+system+and+human+primary+immunodeficiency.">Zebrafish as a model for understanding the evolution of the vertebrate immune system and human primary immunodeficiency.</a> Exp Hematol. 42 (8), 697-706 (2014).</li><li>Babin, P. J., Goizet, C., Raldua, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+of+human+motor+neuron+diseases:+advantages+and+limitations.">Zebrafish models of human motor neuron diseases: advantages and limitations.</a> Prog Neurobiol. 118, 36-58 (2014).</li><li>Seth, A., Stemple, D. L., Barroso, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+use+of+zebrafish+to+model+metabolic+disease.">The emerging use of zebrafish to model metabolic disease.</a> Dis Mod Mech. 6 (5), 1080-1088 (2013).</li><li>Eames, S. C., Philipson, L. H., Prince, V. E., Kinkel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sugar+measurement+in+zebrafish+reveals+dynamics+of+glucose+homeostasis.">Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis.</a> Zebrafish. 7 (2), 205-213 (2010).</li><li>Moss, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+pancreas+in+adult+zebrafish.">Regeneration of the pancreas in adult zebrafish.</a> Diabetes. 58 (8), 1844-1851 (2009).</li><li>Oka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet-induced+obesity+in+zebrafish+shares+common+pathophysiological+pathways+with+mammalian+obesity.">Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity.</a> BMC Physiol. 10 (21), (2010).</li><li>Chu, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Akt1+enhances+adipogenesis+and+leads+to+lipoma+formation+in+zebrafish.">Overexpression of Akt1 enhances adipogenesis and leads to lipoma formation in zebrafish.</a> PLoS One. 7 (5), e36474 (2012).</li><li>Velasco-Santamar&#237;a, Y. M., Korsgaard, B., Madsen, S. S., Bjerregaard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bezafibrate,+a+lipid-lowering+pharmaceutical,+as+a+potential+endocrine+disruptor+in+male+zebrafish+(Danio+rerio).">Bezafibrate, a lipid-lowering pharmaceutical, as a potential endocrine disruptor in male zebrafish (Danio rerio).</a> Aquat Toxicol. 105 (1-2), 107-118 (2011).</li><li>Zang, L., Shimada, Y., Nishimura, Y., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+reliable+method+for+repeated+blood+collection+from+aquarium+fish.">A novel, reliable method for repeated blood collection from aquarium fish.</a> Zebrafish. 10 (3), 425-432 (2013).</li><li>Carmichael, C., Westerfield, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+and+in+vitro+fertilization+at+the+zebrafish+international+resource+center.">Cryopreservation and in vitro fertilization at the zebrafish international resource center.</a> Methods Mol Biol. 546, 45-65 (2009).</li><li>Thorson, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+partitioning+of+body+water+in+Osteichthyes:+phylogenetic+and+ecological+implications+in+aquatic+vertebrates.">The partitioning of body water in Osteichthyes: phylogenetic and ecological implications in aquatic vertebrates.</a> Biol Bull-US. 120, 238-254 (1961).</li><li>Conte, F. P., Wagner, H. H., Harris, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+blood+volume+in+the+fish+(Salmo+gairdneri+gairdneri).">Measurement of blood volume in the fish (Salmo gairdneri gairdneri).</a> Am J Physiol. 205, 533-540 (1963).</li><li>Diehl, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood,+including+routes+and+volumes.">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> J Appl Toxicol. 21 (1), 15-23 (2001).</li><li>Nahas, K., Provost, J. -. P., Baneux, P. H., Rabemampianina, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+blood+removal+via+the+sublingual+vein+on+haematological+and+clinical+parameters+in+Sprague-Dawley+rats.">Effects of acute blood removal via the sublingual vein on haematological and clinical parameters in Sprague-Dawley rats.</a> Lab Anim. 34 (4), 362-371 (2000).</li><li>Curado, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+targeted+cell+ablation+in+zebrafish:+a+new+tool+for+regeneration+studies.">Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies.</a> DevDyn. 236 (4), 1025-1035 (2007).</li><li>Andersson, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+signaling+promotes+regeneration+of+pancreatic+beta+cells+in+vivo.">Adenosine signaling promotes regeneration of pancreatic beta cells in vivo.</a> Cell Metab. 15 (6), 885-894 (2012).</li><li>Hiramitsu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eriocitrin+ameliorates+diet-induced+hepatic+steatosis+with+activation+of+mitochondrial+biogenesis.">Eriocitrin ameliorates diet-induced hepatic steatosis with activation of mitochondrial biogenesis.</a> Sci Rep-UK. 4, 3708 (2014).</li><li>Zang, L., Shimada, Y., Kawajiri, J., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Yuzu+(Citrus+junos+Siebold+ex+Tanaka)+peel+on+the+diet-induced+obesity+in+a+zebrafish+model.">Effects of Yuzu (Citrus junos Siebold ex Tanaka) peel on the diet-induced obesity in a zebrafish model.</a> J Funct Foods. 10, 499-510 (2014).</li><li>Schlegel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+non-alcoholic+fatty+liver+disease+with+zebrafish:+a+confluence+of+optics,+genetics,+and+physiology.">Studying non-alcoholic fatty liver disease with zebrafish: a confluence of optics, genetics, and physiology.</a> Cell Mol Life Sci. 69 (23), 3953-3961 (2012).</li><li>Stoletov, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+lipid+accumulation,+lipoprotein+oxidation,+and+macrophage+lipid+uptake+in+hypercholesterolemic+zebrafish.">Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake in hypercholesterolemic zebrafish.</a> Circ Res. 104 (8), 952-960 (2009).</li><li>Thomas, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrient+balance+and+energy+expenditure+during+ad+libitum+feeding+of+high-fat+and+high-carbohydrate+diets+in+humans.">Nutrient balance and energy expenditure during ad libitum feeding of high-fat and high-carbohydrate diets in humans.</a> Am J Clin Nutr. 55 (5), 934-942 (1992).</li></ol>

The authors have nothing to disclose.

我们在座的详细协议，用于连续获得血液从成年斑马鱼。这种方法是简单的进行，我们使用它在实验室，每天。此采血方法是基于插入玻璃毛细管针进入斑马鱼的背主动脉。在此过程中，关键是要小心不烧蚀脊柱，因为它是标准用于搜索背主动脉。降低颈椎损伤会提高患者的生存率。虽然这种技术是简单，易于掌握，有可以保证高的成功率和存活率的最佳做法。一个熟练的研究员将需要1-2分钟进?...

斑马鱼正日益普及人类疾病的一种有价值的模式，因为他们的器官和遗传学类似于人类1,2。在发育生物学的领域中，许多研究已经证明，斑马鱼和人显示标记相似造血3，止血4,5，和髓6。成年斑马鱼也用于研究免疫学7，神经变性8和肥胖相关疾病9，因为此模式生物股与破坏在人类疾病共同通路。肥胖和肥胖相关疾病（糖尿病，脂肪肝和酒精性脂肪性肝炎和动脉粥样硬化），斑马鱼血糖和血脂水平已经在几个转基因和饮食诱导的肥胖模型10月13日被彻底调查。 从单个动物反复采血会减少动物的使用和DECrease个体差异。然而，重复的样品采集是因为它们具有相对小的血容量和缺乏方便船舶在小动物，如斑马鱼在技术上困难。已经开发了几种方法用于从斑马鱼一次性采血，虽然这些方法有其自身的缺陷，包括致命性，相关的组织损伤和有限的血容量。例如，1-5微升血液可以从约0.3厘米的横向切口，在背主动脉5的区域收获长度。斩首用剪刀通过肩带切割可收集5-10微升血液10。另一种方便的采血方法是尾切除14。心脏穿刺是用于从相同的鱼重复采血一个潜在的替代方法，但非常小的量，得到（约50 NL）与此过程限制的分析，可以是perfor的数量配有11。因此，新的协议需要启用反复的非致命的血液采样，这将是一个重要的进步所必需的这种微生物是一个标准的模式生物的人类疾病。这种技术将允许用于测试药理反应，发现分子生物标记物的诊断，确定预后，以及各种疾病，如代谢疾病，退行性疾病和多种类型的恶性肿瘤的监测。 因此，我们开发了获得血液从斑马鱼连续15微创方法。在这里，我们演示程序可视化，并提供该技术的详细协议。使用这种方法，基于各种参数，包括血红蛋白，空腹血糖，并在健康成年斑马鱼的血脂正常值进行了评价。此外，我们还评估了此方法是否适用于需要通过米系列样本的研究期间过度喂食实验onitoring中血糖水平的瞬时变化。

<table><tbody><tr><td>Glass capillaries with filament</td><td>Narishige</td><td>GD-1</td><td>1.0 mm outer diameter.</td></tr><tr><td>Needle puller</td><td>Narishige</td><td>PC-10</td><td>To produce the needles</td></tr><tr><td>Heparin</td><td>Wako Pure Chemical Industries</td><td>081-00136</td><td>For heparinization</td></tr><tr><td>Aspirator tube assembly</td><td>Drummond</td><td>2-040-000</td><td>For blood collection</td></tr><tr><td>Bulb dispenser</td><td>Drummond</td><td>1-000-9000</td><td>For blood collection</td></tr><tr><td>2-phenoxyethanol</td><td>Wako Pure Chemical Industries</td><td>163-12075</td><td>For anesthetizing the fish</td></tr><tr><td>DRI-CHEM3500V</td><td>Fujifilm</td><td>-</td><td>For hemoglobin measurement</td></tr><tr><td>DRI-CHEM Slides</td><td>Fujifilm</td><td>Hb-WII</td><td>For hemoglobin measurement</td></tr><tr><td>Glutest Neo Super</td><td>Sanwa Kagaku Kenkyusho</td><td>-</td><td>For bood glucose measurement</td></tr><tr><td>Wako L-type TG kit</td><td>Wako Pure Chemical Industries</td><td>464-44201</td><td>For TG measurement</td></tr><tr><td>Wako L-type CHO kit</td><td>Wako Pure Chemical Industries</td><td>460-44301</td><td>For total cholesterol measurement</td></tr><tr><td>Parafilm M</td><td>Alcan Packaging</td><td>PM996</td><td>To expel the blood on</td></tr></tbody></table>

repeated blood collection for blood tests in adult zebrafish

重复采血是实验室动物进行的最普遍的技术之一。但是，还没有建立一个非致命性协议的采血从斑马鱼。以前的方法采血从斑马鱼是致命的，如外侧切口，斩首和尾巴切除。因此，我们已经开发了一种新的"重复"的血液采集方法，以及在座的详细协议，概述了此过程。这种方法是微创并导致非常低死亡率（2.3％），用于斑马鱼，从而使来自同一个体的重复采血。血液取样的最大体积是依赖于体重的鱼。间隔重复采集血样的体积应每周体重或≤1％，每2周，这是由血液中的血红蛋白的测定评价≤0.4％。此外，血红蛋白，空腹血糖，血浆甘油三酯（TG）和总碳在男性和女性的成年斑马鱼holesterol水平进行了测量。我们也应用这个方法来研究糖代谢饮食诱导的肥胖的失调。这种血液采集方法将允许许多应用，包括葡萄糖和脂质代谢和血液学研究，这将增加使用斑马鱼作为人类疾病模型有机体。

该采血方法导致最少伤害的斑马鱼（A &lt;1毫米穿刺; 图1J），并产生一个非常低的死亡率为2.3％。我们研究了血液，可以从一个单一的鱼进行收集和评估的关系，它的体重（图3）的最大容积。我们发现，收集到的最大的血容量呈线性体重（R = 0.813）相关。的血液从个体鱼（体重=1.071克）收集到的最大体积为25微升，并最小体积是从鱼体重0.115克1.3微升。这表明，血液收集?...

Watch this Scientific Journal Video about Repeated Blood Collection for Blood Tests in Adult Zebrafish at JoVE.com

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

重复采血车在成年斑马鱼验血

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

repeated-blood-collection-for-blood-tests-in-adult-zebrafish

Research

JoVE Journal

Biology

34.9K 次观看.  Mie University. Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

视频: Repeated Blood Collection for Blood Tests in Adult Zebrafish

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

科研

遗传学

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

在成年斑马鱼的眼窝射出

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

生物学

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

使用流动细胞测量来检测和量化正在发育的斑马鱼中改变的血液形成

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

发育生物学

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

检查斑马鱼肌肉疾病模型中的肌肉再生

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

成年斑马鱼的优化静脉注射

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in ...

癌症研究

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

在成年斑马鱼的生化分析采血车

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

通过鱼眼研究糖尿病: 成人 tg (fli: EGFP) 斑马鱼视网膜血管的显微解剖、可视化和分析

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

医学

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging成年斑马鱼

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

在 Vivo 表面心电图中成人斑马鱼

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...