Name: simple and effective administration and visualization of microparticles in the circulatory system of small fishes using kidney injection
Uploaded: 2018-06-17T15:00:00.000+00:00
Duration: 13 min 28 s
Description: Watch this Scientific Journal Video about Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection at JoVE.com

作者非常感谢伯登 Osadchiy 和叶甫 Protasov (伊尔库茨克州立大学) 在编写视频协议方面的帮助。这项研究得到了俄罗斯科学基金会 (#15 14-10008) 和俄罗斯基础研究基金会 (#15-29-01003) 的支持。

所有实验程序都是按照欧盟 2010/63/欧盟关于动物实验的指令进行的, 并经伊尔库茨克州立大学生物研究所动物学科研究委员会批准。
1. 微胶囊的制备
注: 携带荧光染料的微胶囊采用相反带电聚电解质7、8的逐层组装制备。所有的程序都是在室温下进行的。
<ol><li>为了合成多孔碳酸钙3 microcores 包围荧光染料, 混合2毫升的 SNARF-1-dextran 溶液 (大多数聚合物绑定荧光染料, 如 FITC-BSA 可以使用) 在2毫克/毫升浓度约0.6 毫升每1摩尔/L 溶液 CaCl2和 Na2CO3下快速搅拌。 注意: 注意荧光染料对漂白的不同敏感性;如果使用光敏荧光探针 (如 SNARF-1), 则必须以尽可能少的光来执行微粒的操作和储存。</li>
	<li>经过5-10 秒的搅拌, 将悬浮液转移到2毫升离心管和离心机, 十五年代在1000万克碳酸钙3 microcores。</li>
	<li>丢弃上清, 用〜2毫升的去离子水洗涤芯, 并通过摇动并用重悬颗粒。</li>
	<li>重复离心洗涤程序总共三次。最后一次离心后, 丢弃上清。</li>
	<li>在超声波浴中孵化 microcores 1 分钟以减少它们的聚集。 注意: 不要忘记用耳机保护耳朵。</li>
	<li>为了将第一个聚合物层存放在模板上, 并用重悬在1摩尔/升氯化钠中, 将4毫克/毫升溶液中的2毫升的核 (烯丙胺盐酸盐) (多环芳烃)。<ol>
<li>在溶液中保持 microcores 5 分钟, 持续晃动。</li>
		<li>十五年代离心后, 用未结环芳烃丢弃上清液。通过多次离心和洗涤步骤, 将覆盖的 microcores 用去离子水清洗至少3次。最后一次离心后, 丢弃上清。</li>
		<li>在超声波浴中孵化 microcores 1 分钟以减少它们的聚集。 注: 如果应用的荧光染料是阳离子, 从聚 (4-苯乙烯) (PSS) 开始在1摩尔/升氯化钠 (见步骤 1.7)。</li>
	</ol>
</li>
	<li>重复步骤 1.6, 用2毫升4毫克/毫升的 PSS 溶液 (也含有1摩尔/升氯化钠) 将第二个聚合物层存入模板。</li>
	<li>重复步骤1.6 和1.7 六次, 以存入12聚合物层。 注意: 建议不要在程序中进行长时间的休息 (12 小时或以上), 直到3-5 层沉积, 因为碳酸钙3 microcores 没有覆盖可能会结晶。请注意, PSS 覆盖率会导致 microcores 的更高聚合, 只有当用聚乙二醇 (PLL) 接枝的多环芳烃或聚 l-赖氨酸是外层层时, 才可取长时间停顿。</li>
	<li>孵化的覆盖 microcores 2 毫克/毫升锁相环-g PEG (1 毫升每微细) 至少2小时。<ol>
<li>通过顺序离心和泥沙步骤冲洗 microcores 水。最后一次离心后, 丢弃上清。</li>
	</ol>
</li>
	<li>要获得中空微囊, 通过加入2毫升0.1 摩尔/升乙二胺四乙酸 (EDTA) 溶液 (经调整为 pH 7.1 和氢氧化钠), 将碳酸钙3模板分解为覆盖的 microcores。<ol>
<li>经过5分钟的孵化, 离心的微囊四十五年代, 并放弃上清与 EDTA。</li>
		<li>重复步骤 1.10-1. 10.1 两次。</li>
	</ol>
</li>
	<li>在四十五年代内通过多次离心步骤清洗微囊, 0.9% 氯化钠三次, 然后洗涤步骤。最后的离心步骤后, 丢弃上清。 注: 注射用的最终微胶囊溶液必须保持不育 (例如, 加入氨苄西林, 0.1 毫克/毫升), 介质应与调查对象进行生物相容性 (与中性 pH 值的等渗介质)。</li>
	<li>在荧光显微镜下估计 hemocytometer 中制备的微囊的浓度。采用 ImageJ9或等效软件, 对微囊进行了一系列的图像测量, 测定了约100微囊的直径, 并利用直方图对尺寸分布进行了研究。</li>
	<li>在黑暗中存储得到的封装探针。 注: 在不育0.9% 氯化钠的几次洗涤后, 微胶囊可以储存数月到4摄氏度。不推荐在贮存期间完全干燥微胶囊。</li>
</ol>2. 微胶囊化 SNARF-1 的光学设置和标定方法的制备
注: 微胶囊化 SNARF-1 的粗糙 pH 值可以使用荧光显微镜7的两个通道中的图像进行, 但在本协议中应用了连接到光纤光谱仪的单通道荧光显微镜。
<ol><li>根据所应用的荧光染料的特点, 将所需的荧光过滤器放在荧光显微镜上, 并打开荧光灯。<ol>
<li>把杠杆拉出目镜。 注意: 过量的光线会破坏光谱仪矩阵。因此, 在不使用光谱仪的情况下, 请确保杠杆处于 "目镜" 模式。</li>
		<li>将光纤的一端连接到光谱仪, 另一端与准直器相连。使用适配器, 将准直器放在照相机管或荧光显微镜的其他可用端口的焦点上。</li>
		<li>打开光谱仪。运行光谱仪控制程序, 并准备光谱仪测量。</li>
	</ol>
</li>
	<li>对于微胶囊批次的校准, 在显微镜滑动处放置5µL 的微胶囊悬浮液 (每µL 10 000 微胶囊), 并在一个黑暗的地方干掉 (例如, 在恒温器35摄氏度)。<ol>
<li>为了校准微胶囊化 SNARF-1 的光谱特性, 在6-9 范围内使用一系列不同 pH 值的缓冲器。将10µL 的缓冲液放到干燥的微胶囊上, 用 SNARF-1-dextran 盖上盖玻片。</li>
		<li>将玻璃滑梯放在显微镜台上。使用×40目标定位微胶囊。</li>
		<li>将显微镜拉杆转向相机端口。用光谱仪注册它们的荧光。把杠杆转回到目镜。 注: 确保光谱信号远超过背景水平, 并确保在视图领域的微胶囊不在气泡 (如果需要切换到较低的放大倍数)。避免同一微胶囊的长时间光照。SNARF-1 对漂白敏感。</li>
		<li>重复步骤2.2.3 不同的微囊10-15 次。</li>
	</ol>
</li>
	<li>计算所有已注册光谱的荧光峰值比值 (例如, 使用 R 或 Scilab), 并使用以下公式确定中值比值 (每个缓冲区) 和中 pH 值之间的回归线: <img alt="Equation 1" src="/files/ftp_upload/57491/57491eq1.jpg"> 注: SNARF-1 具有与质子和 deprotonated 染料的发射相对应的两个峰值的光谱, 峰值之间的比值响应介质的 pH 值。本研究采用605和 660 nm 荧光强度的比值。根据所使用的筛选器集选择这些波长。a和b是由非线性回归 (例如, 使用 R) 确定的系数。值0.15 和1.1 分别是在校准期间观察到的605/i660 的最小和最大值。</li>
	<li>从大约5只成年动物身上收集大约10µL 的鱼血。将鱼放入培养皿中, 用1µL/毫升水悬浮丁香油进行麻醉, 等到动物转向它的一侧, 停止对鳍的捏 (通常是2-3 分钟) 的反应。把鱼转移到玻璃滑梯上。用柳叶刀切断鱼尾, 从尾静脉收集大约2µL 的鱼血。 注: 为防止血液凝固, 用肝素 (5000 U/毫升) 治疗切口, 使用血气玻璃毛细血管和离心管收集血液。<ol>
<li>用吸管将10µL 的血液滴在微电极的尖端上, 用 ph 表确定 ph 值。</li>
		<li>将血液滴到带有干微胶囊的滑梯上, 并按照校准缓冲器 (步骤 2.2-2.3) 的说明, 注册荧光强度的比值。</li>
	</ol>
</li>
	<li>调整校准曲线的线性系数, 使曲线与鱼血中的测量相匹配 (详情见 Borvinskaya等20175)。</li>
</ol>3. 注射准备
<ol><li>用锋利的柳叶刀除去塑料, 从胰岛素笔 (或注射器) 的尖端释放出钢针。 注: 任何细针 (Ø0.33 毫米或更小) 或玻璃毛细管 (通常Ø1毫米) 可以为显微注射10,11准备。</li>
	<li>将针插入玻璃 microcapillary 的一半;使用气体火炬快速和轻轻地焊料。</li>
	<li>将玻璃 microcapillary 连接至 microinjector, 用无菌水冲洗三次。确保液体流经针头。</li>
	<li>用蒸馏水填满系统。 注意: 确保系统中没有气泡。</li>
</ol>4. 注塑
<ol><li>并用重悬微囊的准备悬浮 (在无菌0.9% 氯化钠, 或任何其他用于注射的培养基上, 浓度为0.5 到600万微胶囊每微升) 使用超声波浴1分钟。 注: 由于微囊倾向于沉淀, 在以下注射过程中, 用微胶囊机械 (使用转子) 摇动瓶子, 或手动每隔几分钟并用重悬, 防止它们聚集。</li>
	<li>将鱼放入带有麻醉剂的培养皿中 (在水中悬浮的丁香油0.1 毫升/升) 为 ~ 2-3 分钟. 等待, 直到鱼转向它的一侧, 停止响应的鳍轻捏。</li>
	<li>使用勺子, 把鱼从麻醉剂中转移出来, 轻轻地将它放在一个侧向的潮湿的海绵上, 头部向左 (右撇子) 或向右 (左手的人)。</li>
	<li>就在注射前, 吸1-2 毫米空气进入玻璃毛细管与 microinjector 连接。然后, 加载它与大约2µL 分散微囊。 注: 在注射前, 微胶囊溶液必须调整到保存鱼的温度。</li>
	<li>用非显性手轻轻地将鱼的身体稳定在海绵上。<ol>
<li>找到鱼的侧线。心理上选择从笼盖延伸到腹腔末端的一段。找到这段的中间部分。把针1毫米低的腹部方向。</li>
		<li>用刮擦动作, 轻轻地将鱼鳞放在一旁, 然后进行穿刺。用45°的角度将针头插入到工作台表面。</li>
		<li>把针头推到脊椎上, 直到它仔细地对着它。</li>
		<li>释放大约1µL 的微囊悬浮入肾脏, 慢慢地提取针。 注意: 为了更容易找到合适的穿刺部位, 用底部的光线透照鱼来寻找躯干肾脏是有用的, 如图 2A 和 2B所示。</li>
	</ol>
</li>
	<li>将鱼从头到尾用一条水冲洗, 以除去注射部位溢出的微囊。</li>
</ol>5.在体内可视化
<ol><li>使用解剖剪刀去除鱼头上的鳃盖, 掀鱼鳃。用清水冲洗鳃。</li>
	<li>使用勺子, 把鱼转移到显微镜幻灯片, 并把它放在荧光显微镜的舞台上。 注意: 确保鱼鳃在连续的过程中不干燥。为了避免这种情况, 用巴斯德吸管定期用水 (大约每1-2 分钟) 滋润它们。</li>
	<li>使房间变暗, 使用低放大倍数 (x10 目标) 检查鳃以找到荧光微囊。 注: 当该程序用于将某些荧光微粒引入鱼类循环系统时, 建议在注射前检查几个人的鳃, 以检测意外的荧光微粒。野生斑马鱼的鳃没有自体荧光, 但在某些情况下, 有零星的荧光微粒 (如食物片断或单细胞共生) 可能存在于鳃上。如有必要, 这些粒子可以根据其特定的形状来识别 (例如, 食品件的形状不规则, 不像球形微囊) 或荧光光谱 (即颜色)。<ol>
<li>将透镜切换到更高的幅度 (×40目标), 并将微囊或一组微胶囊放置在视野中心。</li>
		<li>用连接的光谱仪将拉杆转向端口。记录光谱信号。</li>
		<li>把杠杆转回到目镜上。</li>
		<li>对不同的微胶囊重复测量几次。</li>
	</ol>
</li>
	<li>把鱼转到水族馆, 用适当的通气进行恢复。 注意: 使用最少的练习, 可以以每条鱼的大约2-3 分钟的速度进行注射和信号记录。该测量可以重复一个人多次使用重复低, 无害剂量的麻醉或另一种固定方法。对于长期观察, 使用连续麻醉的系统12。</li>
</ol>

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作者没有什么可透露的。

为了证明微粒注入斑马鱼肾脏, 我们使用了半渗透性微胶囊加载的指示染料。因此, 该协议包含使用相反带电聚电解质7、8、15、16、17 的逐层组装制备微囊的指示 ,18 (图 1A)。这项技术的优点是它易于使用现有的实验室设备。根据所使用的条件和化合物, 制备的微囊可从0.5 到100µm, 纳米厚的聚电解质壳15。本手稿中所描述的合成参数导致的弹性微囊包括12层 (除最终生物相容层) 的聚合物大约2-6 µm 的大小 (图 1B和 1C)。决定微囊大小的最重要步骤是模板 microcores 的形成。这一过程涉及碳酸钙晶体的自发形成, 因此, 所得到的粒子是不均匀的。因此, 每个批次都应对微胶囊尺寸分布进行描述。
该协议的重要阶段是在不使用微操作系统技术的情况下, 优化将微粒运入斑马鱼循环系统。对全细胞的复古轨道注射的管理, 以前已经详细描述了3。然而, 根据我们的经验, 新手用户不容易获得作者描述的注射效率, 因为复古眶窦非常小, 位于靠近咽和鳃弓, 这是容易意外伤害用针。由于注射所需的精确度少于毫米, 注射得当是一项相当困难的任务。同样快速和更有效的选择 (表 1) 是直接注射到鱼肾实质。在注射过程中, 针机械地损害肾毛细血管和血管 (例如最大的后根静脉), 允许微粒进入循环系统 (图 2E)。最后, 成年斑马鱼主干肾脏的中央隆起足够大 (可达2毫米), 无需显微外科设备即可手动注射。
正确定位注射针是一个成功的手工管理的关键。你可以通过透照鱼使用底光来练习在完好的动物中找到主干肾脏, 如图 2A和 2B所示。图 2B中的方案演示如何正确执行注入。如果该程序未能管理微粒进入血流, 再注射可以在同一穿刺在短时间内, 几乎没有影响的生存率的个人。
注射到成年斑马鱼的主干肾脏 (也成功地测试在成人的孔雀孔雀鱼柑橘) 是一种有效的方法, 微粒交付到鱼血液与允许的动物死亡率;然而, 由于注射量的变化, 它并不完全适合药物管理。这项技术的一个弱点是由于快速的管理, 从肾脏到腹部的解答的一个重大泄漏。然而, 注射到血液中的物质的相对数量可以用鳃毛细血管的可视化粗略估计 (表 2)。注射量没有严格的限制, 但1多µL 的管理似乎是无效的。最好的玻璃毛细血管可用于微注射;然而, 由于大量的微囊倾向于煽动聚合, 我们建议使用 31-29G (直径0.33-0 毫米 25 mm) 的钢针, 以避免在针腔内堵塞。
通过对鳃中荧光微粒的存在进行快速检查, 可以对微胶囊通过肾脏注入血液中的成功进行监测。鳃是容易接近的器官在体内观察。鳃丝是由一层薄薄的呼吸道上皮覆盖的毛细血管网络, 它使鳃内血液流动的活体成像特别方便 (一种天然的光学窗)。为了进行观察, 软骨鳃盖可以切开以暴露花丝。这种做法是安全的鱼, 它可以生活与裸露的鳃在良好的加气水没有减少预期寿命。此外, 大鱼的鳃可以通过显微镜检查, 只需把笼盖的方式推到19。如果注射成功, 荧光微粒立即出现在鳃中 (图 3)。注意, 植入微粒在循环血容量中有相当大的溶解, 颗粒数量减少到鳃毛细血管, 因此在鱼鳃中应选择足够的微粒浓度来检测注射入鱼肾 (表 2)。
建议的植入程序可应用于涉及不同种类小鱼的广泛研究。尽管该技术已开发和优化注入荧光微粒进入循环系统, 也可用于植入非有色微/纳米微粒或溶解物质;然而, 在这种情况下, 注射的有效性应以其他方式加以验证。目前所描述的步骤是最佳的目的, 如生理监测使用不同的光学微或纳米传感器, 可视化的血管, 提供疫苗或药物的一些光学可见的载体, 并植入基因改良细胞。

在药物和疫苗的提供1、血管可视化2、转基因细胞植入3和微型光学传感器植入等领域, 微粒径颗粒的管理是一项重要任务。4,5. 然而, 微型微粒进入小实验动物的血管系统的植入程序是困难的, 特别是对于微妙的水生生物。对于像斑马鱼这样的热门研究标本, 建议使用视频协议来澄清这些程序。
心内和毛细血管 microinjections 需要训练有素的人员和独特的显微外科设施, 以提供 microobjects 到斑马鱼的血液。此前,3是一种简便有效的全细胞管理方法。然而, 在我们的经验中, 由于眼毛管网络的面积很小, 这项技术需要大量的实践才能达到预期的效果。
在此, 我们描述了一种方法, 通过手动注射直接注入到富含毛细血管和肾血管的成年斑马鱼的肾脏组织中, 以强力有效的微粒注入循环系统。该技术是基于视频协议的细胞移植到斑马鱼肾脏6, 但创伤和耗时的显微手术步骤被淘汰。该方法具有低死亡率 (0-20%) 和高效率 (70-90% 成功) 的特点, 易于使用常用设备。
建议的协议的一个重要部分是可视化的植入微粒 (如果它们是荧光或彩色) 在鳃毛细血管, 这允许验证注射质量, 一个粗略的相对评估的数量注入粒子, 并检测光谱信号的生理测量直接从循环血液。作为描述技术的可能应用的一个例子, 我们展示了在活体测量斑马鱼血液 pH 值使用微胶囊化荧光探针, SNARF-1, 最初建议在 Borvinskaya 等.20175。

<table><tbody><tr><td>SNARF-1-dextran, 70000 MW</td><td>Thermo Fisher Scientific</td><td>D3304</td><td>Fluorescent probe. Any other appropriate polymer-bound fluorescent dye can be used as a microcapsule filler</td></tr><tr><td>Albumin-fluorescein isothiocyanate conjugate (FITC-BSA)</td><td>SIGMA</td><td>A9771</td><td>Fluorescent probe</td></tr><tr><td>Rhodamine B isothiocyanate-Dextran (RITC-dextran)</td><td>SIGMA</td><td>R9379</td><td>Fluorescent probe</td></tr><tr><td>Calcium chloride</td><td>SIGMA</td><td>C1016</td><td>CaCO&lt;sub&gt;3&lt;/sub&gt; templates formation</td></tr><tr><td>Sodium carbonate</td><td>SIGMA</td><td>S7795</td><td>CaCO&lt;sub&gt;3&lt;/sub&gt; templates formation</td></tr><tr><td>Poly(allylamine hydrochloride), MW 50000 (PAH)</td><td>SIGMA</td><td>283215</td><td>Cationic polymer</td></tr><tr><td>Poly(sodium 4-styrenesulfonate), MW 70000 (PSS)</td><td>SIGMA</td><td>243051</td><td>Anionic polymer</td></tr><tr><td>Poly-L-lysine [20 kDa] grafted with polyethylene glycol [5 kDa], g = 3.0 to 4.5 (PLL-g-PEG)</td><td>SuSoS</td><td>PLL(20)-g[3.5]-PEG(5)</td><td>Final polymer to increase the biocompatibility of microcapsules</td></tr><tr><td>Sodium chloride</td><td>SIGMA</td><td>S8776</td><td>To dissolve applied polymers</td></tr><tr><td>Water Purification System</td><td>Millipore</td><td>SIMSV0000</td><td>To prepare deionized water</td></tr><tr><td>Magnetic stirrer</td><td>Stegler</td><td></td><td>For CaCO&lt;sub&gt;3&lt;/sub&gt; templates formation</td></tr><tr><td>Eppendorf&amp;nbsp;Research&amp;nbsp;plus pipette, 1000 &amp;micro;L</td><td>Eppendorf</td><td></td><td>Dosing solutions</td></tr><tr><td>Eppendorf&amp;nbsp;Research&amp;nbsp;plus pipette, 10 &amp;micro;L</td><td>Eppendorf</td><td></td><td>Dosing solutions</td></tr><tr><td>Pipette tips, volume range 200 to 1000 &amp;micro;L</td><td>F.L. Medical</td><td>28093</td><td>Dosing solutions</td></tr><tr><td>Pipette tips, volume range 0.1-10&amp;nbsp;&amp;mu;L</td><td>Eppendorf</td><td>Z640069</td><td>Dosing solutions</td></tr><tr><td>Mini-centrifuge Microspin 12, High-speed</td><td>BioSan</td><td></td><td>For microcapsule centrifugation-washing procedure</td></tr><tr><td>Microcentrifuge tubes, 2 mL</td><td>Eppendorf</td><td>Z666513</td><td>Microcapsule synthesis and storage</td></tr><tr><td>Shaker Intelli-mixer RM-1L</td><td>ELMY Ltd.</td><td></td><td>To reduce microcapsule aggregation</td></tr><tr><td>Ultrasonic cleaner</td><td></td><td></td><td>To reduce microcapsule aggregation</td></tr><tr><td>Head phones&amp;nbsp;</td><td></td><td></td><td>To protect ears from ultrasound</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>SIGMA</td><td>EDS</td><td>To dissolve the CaCO&lt;sub&gt;3&lt;/sub&gt; templates</td></tr><tr><td>Monosodium phosphate</td><td>SIGMA</td><td>S9638</td><td>Preparation of pH buffers</td></tr><tr><td>Disodium phosphate</td><td>SIGMA</td><td>S9390</td><td>Preparation of pH buffers</td></tr><tr><td>Sodium hydroxide</td><td>SIGMA</td><td>S8045</td><td>To adjust the pH of the EDTA solution and buffers</td></tr><tr><td>Thermostat chamber</td><td></td><td></td><td>To dry microcapsules on glass slide</td></tr><tr><td>Hemocytometer blood cell count chamber</td><td></td><td></td><td>To investigate the size distribution and concentration of the prepared microcapsules</td></tr><tr><td>Fluorescent microscope Mikmed 2</td><td>LOMO</td><td></td><td>In vivo visualization of microcapsules in fish blood</td></tr><tr><td>Set of fluorescent filters for SNARF-1 (should be chosen depending on the microscope model; example is provided)</td><td>Chroma</td><td>79010</td><td>Visualization of microcapsules with fluorescent probes</td></tr><tr><td>Fiber spectrometer QE Pro</td><td>Ocean Optics</td><td></td><td>Calibration of microcapsules under microscope</td></tr><tr><td>Optical fiber QP400-2-VIS NIR, 400 &amp;mu;m, 2 m</td><td>Ocean Optics</td><td></td><td>To connect spectrometer with microscope port</td></tr><tr><td>Collimator F280SMA-A</td><td>Thorlabs</td><td></td><td>To connect spectrometer with microscope port</td></tr><tr><td>Glass microscope slide</td><td>Fisherbrand</td><td>12-550-A3</td><td>Calibration of microcapsules under microscope</td></tr><tr><td>Coverslips, 22 x 22 mm</td><td>Pearl</td><td>MS-SLIDCV</td><td>Calibration of microcapsules under microscope</td></tr><tr><td>Glass microcapillaries Intra MARK, 10 &amp;micro;L</td><td>Blaubrand</td><td>BR708709</td><td>To collect fish blood</td></tr><tr><td>Clove oil</td><td>SIGMA</td><td>C8392</td><td>Fish anesthesia</td></tr><tr><td>Lancet No 11</td><td>Apexmed international B.V.</td><td>P00588</td><td>To cut the fish tail and release the steel needle from the tip of insulin autoinjector</td></tr><tr><td>Heparin, 5000 U/mL</td><td>Calbiochem</td><td>L6510-BC</td><td>For treating all surfaces that come in contact with fish blood during fish blood collection</td></tr><tr><td>Seven 2 Go Pro pH-meter with a microelectrode</td><td>Mettler Toledo</td><td></td><td>To determine fish blood pH</td></tr><tr><td>Insulin pen needles Micro-Fine Plus, 0.25 x 5 mm</td><td>Becton, Dickinson and Company</td><td></td><td>For injection procedure. Any thin needle (&amp;Oslash; 0.33 mm or less) is appropriate</td></tr><tr><td>Glass capillaries, 1 x 75 mm</td><td>Hirschmann Laborger&amp;auml;te GmbH &amp;amp; Co</td><td>9201075</td><td>For injection procedure</td></tr><tr><td>Gas torch</td><td></td><td></td><td>To solder steel needle to glass capillary</td></tr><tr><td>Microinjector IM-9B</td><td>NARISHIGE</td><td></td><td>For precise dosing of microcapsules suspension</td></tr><tr><td>Petri dishes, 60 mm x 15 mm, polystyrene</td><td>SIGMA</td><td>P5481</td><td>For manipulations with fish under anesthesia</td></tr><tr><td>Plastic spoon</td><td></td><td></td><td>For manipulations with fish under anesthesia</td></tr><tr><td>Damp sponge</td><td></td><td></td><td>For manipulations with fish under anesthesia</td></tr><tr><td>Dissection scissors</td><td>Thermo Scientific</td><td>31212</td><td>To remove the gill cover from the fish head</td></tr><tr><td>Pasteur pipette, 3.5 mL</td><td>BRAND</td><td>Z331767</td><td>To moisten fish gills</td></tr></tbody></table>

simple and effective administration and visualization of microparticles in the circulatory system of small fishes using kidney injection

将微粒径粒子系统地管理到生物体中, 可用于血管可视化、药物和疫苗的提供、转基因细胞的植入和微小的光学传感器。然而, 静脉注射 microinjections 的小动物, 主要用于生物和兽医实验室, 是非常困难的, 需要训练有素的人员。在此, 我们展示了一种健壮和有效的方法, 引入微粒进入成年斑马鱼 (斑马斑马) 的循环系统, 注入到鱼肾。为了对微血管中引入的微粒进行可视化, 我们提出了一种简单的鱼鳃活体成像技术。在体内监测斑马鱼的血液 pH 值是使用注射微胶囊荧光探针, SNARF-1, 以证明其中一个可能的应用的描述技术。本文详细介绍了 pH 敏感染料的封装, 并说明了该微胶囊在荧光信号活体记录中快速注射和可视化的原理。所提出的注射方法的特点是低死亡率 (0-20%) 和高效率 (70-90% 成功), 并易于使用常用设备。所有被描述的规程可以执行对其他小鱼种类, 例如孔雀鱼和鳉。

得到的结果来自三个主要类别的协议: 荧光染料的封装形成荧光微粒 (图 1), 肾脏注射微囊的进一步可视化鳃毛细血管 (图 2 和 3), 最后,在体内光谱记录 SNARF-1 荧光监测血液 pH 值 (图 4)。
逐层方法使用模板碳酸钙3芯的涂层, 将染料由多个相反电荷的聚合物 (多环芳烃和 PSS) 和一个最外层的生物相容性聚合物 (PLL-克-PEG) 包裹在一起, 是一种简单而经济高效的方法。允许封装不同的荧光探针, 如 SNARF-1-dextran, FITC 或其他 (图 1A)。结果, 得到了具有稳定的半透弹性壳体和测微的中空微胶囊 (图 1B)。这种技术制备的微胶囊通常是不均匀的, 在每个批次中均有颗粒大小和特征中值的正态分布 (图 1С)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57491/57491fig1.jpg"> 图 1: 逐层 荧光染料中空聚电解质微囊的合成与表征。(a)微胶囊组装计划 (从 Gurkov等20164发表的类似计划中得出)。(B)载有荧光染料 FITC 的中空微囊的图片。(C) 图1B 所示的批微囊的尺寸表征。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57491/57491fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
在小鱼循环系统中强健有效的微粒注入可以通过手动注射直接注入鱼肾。在鱼体 (躯干肾脏) 的适当部位穿刺对于快速运送微粒的人工注射至关重要。鱼肾是一种造血器官, 含有色素 melanophores, 因此, 它的颜色很好。因为它位于游泳膀胱的透明腔之间, 所以很容易在完好的动物中识别出虚弱的色素沉着, 或者使用底层光源透视小鱼, 如图 2A和 2B所示。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57491/57491fig2.jpg"> 图 2: 正确定位肾脏注射部位。(A)对斑马鱼的反光照显示树干肾脏的定位 (箭头)。(B)计划说明如何识别合适的穿刺地点。白色虚线表示鱼腹段的侧线。箭头指示穿刺部位和向脊柱注射的正确方向。游泳膀胱由绿线指定。(C)在切除器官和体壁后可视化斑马鱼肾脏。箭头指向树干肾脏的中央隆起。(d) 斑马的矢状组织学切片说明了成年鱼内脏的一般解剖(H & E 染色).(E)用箭头指着后根静脉的流明, 从 (D) 缩放的鱼肾脏的显微图像。星号表示游泳膀胱。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57491/57491fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了检查注射过程的成功与否, 在切割鱼鳃盖 (图 3A) 后, 应在低放大率 (x10-20 目标) 下快速对鳃进行目视检查。尽管大多数微囊残留在注射部位或溢出到体腔 (图 3B), 如果注射正确, 可以观察的荧光微囊自由漂浮在鳃毛细血管注射后立即裸露的鱼鳃 (图 3C)。如果鳃中没有发现荧光微粒, 则可以在同一穿刺中重复注射。成功地运送到血液应该在总注射的鱼的大约70-90% 获得。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57491/57491fig3.jpg"> 图 3: 微囊肾注射, 鳃毛细血管进一步可视化. (A)将微胶囊输送到斑马鱼血液中的整体方案。(B)在用绿色 FITC-BSA 染料注射微胶囊后, 斑马鱼的荧光图像 (穿刺部位由箭头表示)。(C)这张鱼鳃的图片, 从 (B) 的比例, 显示了肾注射成功地提供微囊 (野生型斑马鱼的鳃没有自发荧光)。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57491/57491fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
在直接注射到鱼树干肾脏, 广泛的瘀伤通常形成在穿刺部位之下, 表明损伤的实质毛细血管或肾血管。由于穿刺似乎迅速收缩, 身体内没有血漏。尽管内部出血, 个人仍然可以恢复约80-90% 幸存的过程中 (表 1)。还知道, 在伤害13、14之后, 鱼类可以再生肾单位。如果超过20% 的鱼正在死亡, 你必须确保麻醉的正确执行。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td rowspan="2">封装荧光染料</td>
			<td rowspan="2">n</td>
			<td rowspan="2">浓度, 微胶囊每µL</td>
			<td rowspan="2">注入量, µL</td>
			<td rowspan="2">微囊的平均直径, µm</td>
			<td rowspan="2">成功交付入鱼血液,%</td>
			<td colspan="2">注射后死亡率,%</td>
		</tr>
<tr>
<td>1小时</td>
			<td>1天</td>
		</tr>
<tr>
<td colspan="8">射入树干肾脏</td>
		</tr>
<tr>
<td>FITC-BSA</td>
			<td>36</td>
			<td>4 x 106
</td>
			<td>1。6</td>
			<td>2。7</td>
			<td>94</td>
			<td>8</td>
			<td>3</td>
		</tr>
<tr>
<td>SNARF-1-dextran</td>
			<td>29</td>
			<td>3-6 x 105
</td>
			<td>1-2</td>
			<td>5。1</td>
			<td>72</td>
			<td>7</td>
			<td>12</td>
		</tr>
<tr>
<td>RITC-葡聚糖</td>
			<td>20</td>
			<td>6 x 106
</td>
			<td>2</td>
			<td>2。0</td>
			<td>70</td>
			<td>0</td>
			<td>0</td>
		</tr>
<tr>
<td>0.9% 氯化钠</td>
			<td>41</td>
			<td>-</td>
			<td>1-2</td>
			<td>-</td>
			<td>-</td>
			<td>5</td>
			<td>0</td>
		</tr>
<tr>
<td colspan="8">复古轨道注射</td>
		</tr>
<tr>
<td>FITC-BSA</td>
			<td>9</td>
			<td>1 x 105
</td>
			<td>2</td>
			<td>4。2</td>
			<td>11</td>
			<td>22</td>
			<td>0</td>
		</tr>
<tr>
<td>RITC-葡聚糖</td>
			<td>11</td>
			<td>6 x 106
</td>
			<td>1</td>
			<td>2。0</td>
			<td>9</td>
			<td>18</td>
			<td>0</td>
		</tr>
</tbody></table>表1。将微囊输送到斑马鱼血液中的安全性和效率.过程的成功由荧光材料的存在决定在鱼鳃毛细血管。
如果注入微粒的浓度不足, 可能会有太少的微粒在鱼鳃中快速可视化。同时, 浓度过高的悬浮液会堵塞针头。在逐层组装的微胶囊中, 直径为2-5 µm, 浓度约 4 * 105-6 * 106微胶囊每µL 是最佳的, 以轻松检测鱼鳃后, 注射入鱼肾 (表2)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>浓度, 微胶囊每µL</td>
			<td colspan="7">微囊的数量在显微镜视野 (×20目标) 在不同的个体的鳃 (n = 7 每集中)</td>
		</tr>
<tr>
<td>4 x 106
</td>
			<td>> 100</td>
			<td>> 100</td>
			<td>0</td>
			<td>> 100</td>
			<td>≈70</td>
			<td>≈50</td>
			<td>> 100</td>
		</tr>
<tr>
<td>4 x 105
</td>
			<td>≈10</td>
			<td>≈20</td>
			<td>0</td>
			<td>0</td>
			<td>≈20</td>
			<td>≈40</td>
			<td>≈15</td>
		</tr>
<tr>
<td>4 x 104
</td>
			<td>0</td>
			<td>3</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>4</td>
		</tr>
<tr>
<td>4 x 103
</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr>
</tbody></table>表2。注射后斑马鳃中 FITC-BSA 微囊的视觉计数记录 (1.6 µL) 进入主干肾脏.
采用微胶囊荧光探针、SNARF-1 (图 4) 对斑马鱼血液 pH 值进行体内监测, 提出了将微粒植入鱼类循环系统的方法。SNARF-1 具有与质子和 deprotonated 染料的发射相对应的两个峰值的光谱 (图 4A), 因此可以绘制不同 pH 值之间的峰值比值的校准曲线 (图 4B)。血液的组分影响被封装的 SNARF-1 的读出 (图 4B), 可以通过实验性地评估通过同时测量血液 pH 值用微胶囊和 ph 表与玻璃电极。对斑马鱼血液中微囊的标定曲线, 应按实验测量的 pH 值差的系数改变缓冲曲线 (见 Borvinskaya等20175 )。
在注射微囊后至少几个小时内, 斑马鱼鳃毛细血管的血液 pH 值保持稳定 (图 4C)。同时, 短高碳酸暴露导致血液 pH 值显著降低, 这表明该方法在小鱼体内研究中的适用性。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57491/57491fig4.jpg"> 图 4: 监测斑马鱼血液的典型例子рН通过对微囊化染料 SNARF-1 荧光光谱的注册.(A)在不同 pH 值的磷酸钠缓冲液中 SNARF-1 的制备微囊的光谱。(B)磷酸钠缓冲液中制备的 pH 敏感微囊和提取的斑马鱼血液的校准曲线。对于所有的测量, 描述了平均标准偏差。(C)在斑马鱼鳃毛细血管内用封装荧光 SNARF-1 染料在体内监测斑马鱼血рН的典型例子。在控制条件下, 注射微囊后4小时血液 pH 值保持稳定, 5 分钟暴露在严重高碳酸血症 (900-1000 毫克/升的溶解 CO2) 导致鱼血酸化。星号表示与p < 0.01 (曼-惠特尼 U 测试) 平行对照组的统计学意义差异。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57491/57491fig4large.jpg">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection at JoVE.com

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

小鱼肾注射液循环系统中微粒的简易有效管理与可视化

本文阐述了快速、微创荧光微粒注入小鱼循环系统的原理和鱼血中微粒的体内可视化。

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

simple-effective-administration-visualization-microparticles

Universidad San Sebastian

Research

JoVE Journal

Biology

8.3K 次观看.  Institute of Biology at Irkutsk State University. 本文阐述了快速、微创荧光微粒注入小鱼循环系统的原理和鱼血中微粒的体内可视化。

视频: Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

在成年斑马鱼的全肾骨髓移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

科研

生物学

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

斑马鱼幼虫显微注射和成像的微装置

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

发育生物学

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 ...

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

Intramyocardial Injection for the Study of Cardiac Lymphatic Function in Zebrafish

Zebrafish have proved to be an important model for studying cardiovascular formation and function during postembryonic development and regeneration. The present protocol describes a method for injecting fluorescent tracers into the zebrafish myocardium to study interstitial fluid and debris uptake into cardiac lymphatic vessels. To do so, microspheres (200 nm diameter) and quantum dots (&#60;10 nm diameter) are introduced into the myocardium of live zebrafish, which can be tracked using ex vivo confocal microscopy. These tracers are then tracked intermittently over several hours to follow clearance from the myocardium into cardiac lymphatic vessels. Quantum dots&#160;are transported through cardiac lymphatic vessels away from the heart, while larger microspheres&#160;remain at the injection site for over three weeks. This method of intramyocardial injection can be extended to other uses, including the injection of encapsulated MS or hydrogels to locally release cells, proteins, or compounds of interest to a targeted region of the heart.

The present protocol allows efficient and stable delivery of fluorescent microspheres (MS) and quantum dots (QD) into the myocardium of live fish that can be tracked (traced) over time.

Zebrafish have proved to be an important model for studying cardiovascular formation and function during postembryonic development and regeneration. The ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

直接进入成年斑马鱼的肾细胞移植

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

斑马鱼肾功能评价采用荧光测定间隙

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

微穿刺2光子显微镜辅助小鼠鲍曼空间的研究

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...

Intrathoracic Injection for the Study of Adult Zebrafish Heart

The adult zebrafish heart provides a powerful model in cardiac regeneration research. Although the strength of this system is based on transgenic approaches, a rapid delivery of exogenous factors provides a complementary technique in functional studies. Here, we present a method that relies on administration of a few microliters of solution into the pericardial cavity without causing myocardial damage. Intrathoracic (IT) injections can efficiently deliver proteins and chemical compounds directly onto the heart surface. The injected substances diffuse through the epicardium into the underlying cardiac tissues. Compared to intraperitoneal (IP) injections, the main advantage of intrathoracic injections is the focal administration of the tested factors on the target organ. The delivery of molecules directly into the pericardium is a suitable strategy for studies of cardiac preconditioning and regeneration in adult zebrafish.

This method relies on the injection of 0.5&#8722;3 &#956;L of solution into the thorax of adult zebrafish. The procedure efficiently delivers proteins and chemical compounds into the proximity of the zebrafish heart without damaging the organ. The approach is suitable for testing effects of exogenous factors on various tissues of the heart.