Name: studying diabetes through the eyes of a fish: microdissection, visualization, and analysis of the adult tg(fli:egfp) zebrafish retinal vasculature
Uploaded: 2017-12-26T14:00:00.000+00:00
Duration: 10 min 7 s
Description: Watch this Scientific Journal Video about Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature at JoVE.com

作者想感谢韦克莱斯 Bennewitz 和马琳豪斯纳为斑马鱼饲养和技术援助。作者承认核心设施活细胞成像曼海姆在曼海姆生物医学和医疗技术中心的支持 (DFG 研究所 91027/9-1)。这项研究得到了德意志 Forschungsgemeinschaft (国际研究训练组 1874/1 "DIAMICOM"、项目 SP5 和 SP9 的支持;合作研究中心 SFB1118, 项目 B1 和合作研究中心 SFB/TR23 项目 Z5)。

所有的程序, 包括与动物有关的步骤, 已得到动物伦理委员会 (Regierungspräsidium 卡尔斯鲁厄) 的批准, 并遵循海德堡大学的动物保育指南。
1. 定影剂的制备 (4% 粉煤灰/PBS)
<ol>
	<li>每天准备新鲜的定影液, 以确保组织学组织完整性的最佳保存。</li>
	<li>在室温下不断搅拌, 在90毫升的双蒸馏水 (ddH2O) 中溶解0.2 克氢氧化钠 (氢氧化钠)。</li>
	<li>加入4克甲醛 (粉煤灰) 和搅拌, 直到解决方案是完全明确的至少5-10 分钟, 以确保降解的大分子。 注意: 粉煤灰是有毒的, 小心处理。</li>
	<li>在溶液中溶解0.84 克磷酸二氢钠 (NaH2PO4), 并在7.2 通过测量控制 pH 值。</li>
	<li>使用 ddH2O 将音量调整为100毫升, 并通过3级滤纸过滤解决方案。</li>
	<li>在冰上储存4% 的粉煤灰/PBS。</li>
</ol>
2. 安乐死解决方案的准备
注: 以下步骤与 ABTL 野生型斑马鱼菌株在一般年龄6-8 周一完成。
<ol>
	<li>使用 3-氨基甲烷磺酸乙酯, 也命名为 "tricaine" 或 MS-222, 在0.31 毫克/毫升浓度为斑马鱼安乐死16。利用1x 的卵水, 用来饲养斑马鱼的胚胎, 作为解决安乐死的溶剂。一般情况下, 在使用镇静鱼之前, 要控制正确的麻醉深度, 表现出平衡损失和接触反应损失。<ol>
		<li>达到1000毫升的10x 鸡蛋水, 添加这些盐到1000毫升双蒸馏水 (ddH2O), 同时不断搅拌如下顺序:10 克氯化钠, 0.3 克氯化钾, 0.4 g CaCl2 *6H2o, 1.32 g MgSO4 *6H2o。</li>
	</ol>
	</li>
</ol>
3. 斑马鱼组织的固定
<ol>
	<li>将每样6毫升4% 的粉煤灰/PBS 溶液转移到六井板的井中。</li>
	<li>安乐的成年斑马鱼 (以前保持在一个标准的 light-day 周期,例如, 12 小时:12 h) 2 小时后, 灯打开在早上, 通过放置在 tricaine 的解决方案, 并等待, 直到他们已经达到了安乐死。随着盖运动的停止, 这可能需要10分钟。</li>
	<li>把鱼从 tricaine 的安乐死溶液中取出, 放在新鲜的纸巾上。把鱼弄干, 用手术刀把盖的头砍下来 (参见图 2A)。直接将磁头转移到准备好的板上, 其中含有新鲜的定影剂。</li>
	<li>将鱼头在4° c 过夜至少24小时贮存好, 以确保定影物穿透更深的视网膜层。 注: 斑马鱼头可在4° c 的磷酸缓冲盐水 (PBS) 中以最高 48 h 的浓度储存, 而不需要相应的荧光信号强度损失。延迟的增加会导致组织完整性的丧失和图像质量的降低, 因此应该避免。</li>
</ol>
4. 斑马鱼视网膜血管的制备
<ol>
	<li>用加热的2% 琼脂糖将培养皿填满三分之一, 然后等到琼脂牢固。用 1x PBS 覆盖琼脂板, 创建一个工作区来解剖眼睛。 注意: 这将允许在解剖时保存准备好的镊子和低压力, 减少结构损伤的可能性。所有进一步的准备步骤都应该在这个工作区中使用 #5 在解剖显微镜下的带有细微提示的直钳和额外的 epi 照明。<ol>
		<li>在解剖时, 使用4.0x 和6.0x 之间的放大倍数, 以允许对组织的概述和细节为导向的评估。</li>
	</ol>
	</li>
	<li>将固定的样品转移到培养皿中, 并在切口表面用一镊子, 在眼窝的眼球下方插入另一个钉在一起的镊子。慢慢打开眼睛下面的镊子, 抓住视神经, 然后小心地撕裂和分离眼睛 (图 2B)。</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56674/56674fig2.jpg"> 
图 2: 去除成年斑马鱼眼.角膜侧视图与眼睛仍然在眶腔和完整的视神经(A)。角膜侧视图与分离的眼睛(B)。在此步骤(C)中对斑马鱼眼的示意图描述。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56674/56674fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="3">
	<li>移除仍与眼睛相连的四腹直肌和两个斜眼外肌, 以及将眼睛连接到眼眶的残余眼球组织 (比较图 3和图 4)。<ol>
		<li>通过半镊子的眼睛, 并抓住与其他镊子的结构, 轻轻撕开它与直径运动, 以实现这一点。由于眼球的抓取直接导致内血管破损, 因此该技术是适当的温和保存血管。</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56674/56674fig3.jpg"> 
图 3: 成年斑马鱼眼中眼球外肌的焦点.外侧视野与眼外肌附着在眼球的左外缘在焦点(A)中。视神经侧视与眼球外肌对称侧翼视神经(B)。在此步骤(C)中对斑马鱼眼的示意图描述。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56674/56674fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="4">
	<li>使用一个 27G (0.4 x 19 毫米) 一次性针头刺穿角膜 (图 4C, 红色箭头) 在外面的范围。通过这个开头举行角膜用镊子和轻微地撕毁它打开。之后仔细地工作创造一个中心的泪花近似地各自学生的大小。</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56674/56674fig4.jpg"> 
图 4: 成年斑马鱼眼球切除后的所有眼外肌.侧视野与被清除的外在外缘眼睛地球可见(A)。视神经侧视神经在中部。反光巩膜不覆盖整个区域周围的神经 (红色虚线) (B)。在此步骤(C)中对斑马鱼眼的示意图描述。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56674/56674fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="5">
	<li>在虹膜上方的外角膜边缘上按压眼球 (薤) 的角膜侧。这将创造一个小凹痕, 并推动镜头到高度的角膜撕裂。运行在镜头下的镊子, 并删除它 (图 5A)。</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56674/56674fig5.jpg"> 
图 5: 在去除晶状体的过程中, 成年斑马鱼眼.角膜侧视图与透镜推挤通过角膜泪花(A)。角膜侧视图与透镜在眼睛旁边地球(B)。在此步骤(C)中对斑马鱼眼的示意图描述。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56674/56674fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="6">
	<li>将眼睛倒置到面对研究者的视神经。注意, 巩膜和角膜是相连的, 形成了眼睛的纤维状鳞茎, 以保护杯状的视网膜。(图 6)。<ol>
		<li>这壳, 由角膜和巩膜组成, 也被称为 "加" 和备件周围的视神经 (图 4B, 红色虚线) 的区域。在这个停止处插入一根针, 在巩膜和视网膜之间创建一个开口。</li>
	</ol>
	</li>
</ol>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56674/56674fig6.jpg"> 
图 6: 由巩膜和角膜组成的加, 与其余的眼内组织断开连接.角膜侧视图显示半透明角膜进入色素性巩膜(A)。侧视图集中在加(B)的巩膜部分。已移除加(C)的示意图描述。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56674/56674fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="7">
	<li>使用这两个镊子的访问, 小心地撕裂的巩膜轴向条纹, 以增加周围的视觉神经的开放。在向角膜侧圆周过渡时要注意保持加完好 (请参见图 6A)。</li>
	<li>用一个镊子抓住巩膜, 然后用另一根抓住视神经, 然后拔掉, 完全从眼睛中取出加并丢弃它 (图 6)。在分离加和其余的人工组织之前, 尝试切断任何连接, 因为对其他结构的附着将是一个关键点;这一步将提供一个杯状结构组成的膜和视网膜包含视网膜血管 (图 7)。</li>
	<li>在脉络膜/RPE 层中创建一个破裂, 将27G 针侧向剩余的杯子, 同时用针尖的边缘刮去外表面。使用破裂作为一个接入点, 以获得对脉络膜/RPE 层的控制, 并撕裂它与两个镊子成条纹, 但保持连接到虹膜完整。<ol>
		<li>之后, 运行一个镊子下的虹膜和周围的电路从外部, 而创造紧张通过拉在停止的脉络膜/RPE 层, 以实现脱离的组合结构。 注意: 虹膜必须断开, 以便在可视化容器时不妨碍荧光 (图 8B)。由于虹膜仍然与周围的组织相连, 所以直接抓取虹膜以去除它会导致广泛的血管损伤, 特别是在内视圈 (IOC)。脉络膜层和视网膜色素上皮 (RPE) 可以分离, 并经常保留与虹膜的连接, 这使得容易的二次切除。</li>
		<li>如果虹膜的某些部分不能被移除, 使用一个自然的断裂点成人斑马鱼眼睛展品在感光层 (PL), 以类似的方式4.9 步, 以消除虹膜。</li>
		<li>在剩余的杯状视网膜的外侧刮过, 使停在破裂点上方的 PL (图 11C, 黑色箭头)。使用创建的 access 删除图层的上部, 同时保持与虹膜的可能连接完好无损。然后, 运行在虹膜下的镊子, 并在电路中绕去, 在前面提到的分离的组合结构。 注: 一个人应该锻炼耐心, 因为整个步骤4.9 可能是困难的, 但这种护理将取得更好的血管结果比直接抓住虹膜在边缘的瞳孔或强迫开放通过破坏连接到脉络膜/RPE 或感光层没有各自的去除。因此, 这一步骤将保护视网膜血管的完整性, 但会导致视网膜外层的损伤。</li>
	</ol>
	</li>
</ol>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56674/56674fig7.jpg"> 
图 7: 成年斑马鱼眼切除后的加.侧视图显示残存的眼内组织与完整的虹膜和视神经(A)。焦点(B)中带有虹膜的角膜侧视图。在此步骤(C)中对斑马鱼眼的示意图描述。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56674/56674fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="10">
	<li>使用一个切割2.5 毫米直切边缘的显微切割弹簧剪刀, 以尽可能接近视网膜切断视神经;这将允许一个更好的扁平安装的组织。</li>
</ol>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56674/56674fig8.jpg"> 
图 8: 成年斑马鱼眼球切除后视网膜色素上皮/脉络膜和截光神经.视神经侧视图显示视网膜与截断视神经(a)。角膜侧视图, 直接查看到视网膜的最内层(B)。在此步骤(C)中对斑马鱼眼的示意图描述。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56674/56674fig8large.jpg" target="_blank">请点击这里查看更大版本的这个数字。</a>
5. 视网膜血管的安装
<ol>
	<li>在 1x PBS 中冲洗两次解剖的视网膜5分钟。为了在视神经被截断后转移视网膜, 请使用一个带有微勺末端的实验室刮刀, 以避免直接处理暴露的组织。</li>
	<li>把一滴 PBS 放在玻璃滑梯上, 然后将视网膜转移到水滴中。使用镊子来保持组织的地方, 而切割杯状结构与手术刀创建一个平坦的四花瓣或五花瓣形状取决于视网膜的大小 (请参见图 9)。</li>
	<li>用一张精美的纸把剩下的 PBS 吸干。注意不要触摸视网膜。</li>
	<li>在安装介质上涂上平坦的视网膜, 用片覆盖。注意不要制造泡沫, 因为气泡会扭曲视网膜血管的可视化。</li>
	<li>尽量减少片的运动, 用清晰的指甲油密封盖子。</li>
</ol>
6. 视网膜血管的可视化
<ol>
	<li>尽量缩短准备和可视化之间的时间间隔, 通过荧光信号损耗降低图像细节损耗。存储准备好的视网膜血管在4° c, 以减少信号丢失, 如果直接可视化无法实现, 因为图像质量的退化在准备以后慢慢地变得可看见 48 h。</li>
	<li>可视化视网膜血管支架通过荧光显微镜或激光扫描共聚焦显微镜。 注: 通过荧光显微镜进行血管可视化, 达到2.5x 放大倍数, 曝光时间为2.0 秒, 增益为 6.0x, 伽玛设置为1.67。对于共焦显微镜, Ar 激光器 (488 nm/20 兆瓦) 在20% 的功率是使用与 TD 488/543/633 励磁过滤器, 20 x/0.7 NA multi-immersion 目标与 ddH2O 作为浸入介质, 和排放过滤器设置 505-560 nm。共焦的图片是由 4x4, 4x5, 或5x5 的奇异图像组合, 通过瓷砖扫描, 根据视网膜的大小。</li>
</ol>
7. 斑马鱼视网膜的他部分
注意: 对于斑马鱼视网膜的切片, 按照协议中的描述准备组织, 直到步骤 4.5, 然后直接跳到步骤7.1。
<ol>
	<li>在 1x PBS 中, 洗核眼睛 (4.5 步后) 两次5分钟。然后, 将组织转化为70% 乙醇 (乙醇)。此时, 准备好的眼睛可以储存在4° c, 直到石蜡嵌入。</li>
	<li>在石蜡包埋前, 用以下方法冲洗组织: 2x15 最小在80% 乙醇, 2x15 分钟在90% 乙醇, 3x15 min 在96% 乙醇, 3x15 分钟在99% 乙醇, 2x15 分钟在 acetone/99%ethanol (1:2), 3x15 分钟丙酮。将组织石蜡在62° c 过夜。</li>
	<li>将新鲜的石蜡加热至62° c, 并将其倒入嵌入模具中。直接将组织转移到模具中, 并以快速的速度控制眼睛的正确方向。然后, 将嵌入的盒式纸盒应用到模具上, 然后用额外的加热石蜡盖住。</li>
	<li>在石蜡块冷却后取下嵌入模具。将切片的石蜡块切成10µm 段, 在水面上45° c 的水浴中将其浮在水面上, 使其完全平整。</li>
	<li>在45° c 的烤箱中, 在一夜之间把玻璃片上的部分吸干, 然后垂直排水, 除去多余的水分。</li>
	<li>进一步处理部分与以下日程表对 deparaffinize 和他污点: 4x1 min 在二甲苯, 1x1 min 在99% 乙醇, 1x1 min 在96% 乙醇, 1x1 min 在80% 乙醇, 1x1 min 在70% 乙醇, 1x1 min 在 ddH2O, 1x4 min 在迈尔的苏木精, 1x10 分钟在 ddH2o, 1x2min 在0.5% 曙红, 1x30 s 在 ddH2o, 1x30 在80% 乙醇, 2x30 s 在96% 乙醇, 3x1 min 在99% 乙醇, 1x1 min 在二甲苯。</li>
	<li>把玻璃滑出的二甲苯, 并迅速覆盖的组织与安装媒体。避免让组织完全干燥, 然后在上面放置一个片, 并密封染色组织。</li>
</ol>

<ol>
	<li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Global report on diabetes. , 88 (2016).</li><li>Lim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+nephropathy+-+complications+and+treatment.">Diabetic nephropathy - complications and treatment.</a> Int J Nephrol Renovasc Dis. 7, 361-381 (2014).</li><li>Yau, J. W., 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=Global+prevalence+and+major+risk+factors+of+diabetic+retinopathy.">Global prevalence and major risk factors of diabetic retinopathy.</a> Diabetes Care. 35 (3), 556-564 (2012).</li><li>Cheung, N., Mitchell, P., Wong, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+retinopathy.">Diabetic retinopathy.</a> The Lancet. 376 (9735), 124-136 (2010).</li><li>Raymond, N. 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=Higher+prevalence+of+retinopathy+in+diabetic+patients+of+South+Asian+ethnicity+compared+with+white+Europeans+in+the+community:+a+cross-sectional+study.">Higher prevalence of retinopathy in diabetic patients of South Asian ethnicity compared with white Europeans in the community: a cross-sectional study.</a> Diabetes Care. 32 (3), 410-415 (2009).</li><li>Heng, L. Z., 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=Diabetic+retinopathy:+pathogenesis,+clinical+grading,+management+and+future+developments.">Diabetic retinopathy: pathogenesis, clinical grading, management and future developments.</a> Diabet Med. 30 (6), 640-650 (2013).</li><li>Writing Committee for the Diabetic Retinopathy Clinical Research, N., 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=Panretinal+Photocoagulation+vs+Intravitreous+Ranibizumab+for+Proliferative+Diabetic+Retinopathy:+A+Randomized+Clinical+Trial.">Panretinal Photocoagulation vs Intravitreous Ranibizumab for Proliferative Diabetic Retinopathy: A Randomized Clinical Trial.</a> JAMA. 314 (20), 2137-2146 (2015).</li><li>Jao, L. E., Wente, S. R., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+multiplex+biallelic+zebrafish+genome+editing+using+a+CRISPR+nuclease+system.">Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.</a> Proc Natl Acad Sci U S A. 110 (34), 13904-13909 (2013).</li><li>Sharma, K. R., 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=ELMO1+protects+renal+structure+and+ultrafiltration+in+kidney+development+and+under+diabetic+conditions.">ELMO1 protects renal structure and ultrafiltration in kidney development and under diabetic conditions.</a> Sci Rep. 6, 37172 (2016).</li><li>Baek, J. S., Fang, L., Li, A. C., Miller, Y. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ezetimibe+and+simvastatin+reduce+cholesterol+levels+in+zebrafish+larvae+fed+a+high-cholesterol+diet.">Ezetimibe and simvastatin reduce cholesterol levels in zebrafish larvae fed a high-cholesterol diet.</a> Cholesterol. , 564705 (2012).</li><li>Elo, B., Villano, C. M., Govorko, D., White, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+zebrafish+as+a+model+for+glucose+metabolism:+expression+of+phosphoenolpyruvate+carboxykinase+as+a+marker+for+exposure+to+anti-diabetic+compounds.">Larval zebrafish as a model for glucose metabolism: expression of phosphoenolpyruvate carboxykinase as a marker for exposure to anti-diabetic compounds.</a> J Mol Endocrinol. 38 (4), 433-440 (2007).</li><li>Gut, P., Reischauer, S., Stainier, D. Y. R., Arnaout, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Little+Fish,+Big+Data:+Zebrafish+as+a+Model+for+Cardiovascular+and+Metabolic+Disease.">Little Fish, Big Data: Zebrafish as a Model for Cardiovascular and Metabolic Disease.</a> Physiol Rev. 97 (3), 889-938 (2017).</li><li>Kasprick, D. 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=Microanatomy+of+adult+zebrafish+extraocular+muscles.">Microanatomy of adult zebrafish extraocular muscles.</a> PLoS One. 6 (11), e27095 (2011).</li><li>Gestri, G., Link, B. A., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+visual+system+of+zebrafish+and+its+use+to+model+human+ocular+diseases.">The visual system of zebrafish and its use to model human ocular diseases.</a> Dev Neurobiol. 72 (3), 302-327 (2012).</li><li>Lawson, N. D., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+embryonic+vascular+development+using+transgenic+zebrafish.">In vivo imaging of embryonic vascular development using transgenic zebrafish.</a> Dev Biol. 248 (2), 307-318 (2002).</li><li>Matthews, 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=Anesthesia+and+euthanasia+in+zebrafish.">Anesthesia and euthanasia in zebrafish.</a> ILAR J. 53 (2), 192-204 (2012).</li><li>Bell, B. A., 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=Retinal+vasculature+of+adult+zebrafish:+in+vivo+imaging+using+confocal+scanning+laser+ophthalmoscopy.">Retinal vasculature of adult zebrafish: in vivo imaging using confocal scanning laser ophthalmoscopy.</a> Exp Eye Res. 129, 107-118 (2014).</li><li>Avanesov, A., Malicki, J. <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+the+retina+in+the+zebrafish+model.">Analysis of the retina in the zebrafish model.</a> Methods Cell Biol. 100, 153-204 (2010).</li><li>Gramage, E., Li, J., Hitchcock, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+and+function+of+midkine+in+the+vertebrate+retina.">The expression and function of midkine in the vertebrate retina.</a> Br J Pharmacol. 171 (4), 913-923 (2014).</li><li>Cao, R., Jensen, L. D., Soll, I., Hauptmann, G., Cao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-induced+retinal+angiogenesis+in+zebrafish+as+a+model+to+study+retinopathy.">Hypoxia-induced retinal angiogenesis in zebrafish as a model to study retinopathy.</a> PLoS One. 3 (7), e2748 (2008).</li><li>Jorgens, K., Hillebrands, J. L., Hammes, H. P., Kroll, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+model+for+understanding+diabetic+complications.">Zebrafish: a model for understanding diabetic complications.</a> Exp Clin Endocrinol Diabetes. 120 (4), 186-187 (2012).</li><li>Alvarez, 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=Predominant+cone+photoreceptor+dysfunction+in+a+hyperglycaemic+model+of+non-proliferative+diabetic+retinopathy.">Predominant cone photoreceptor dysfunction in a hyperglycaemic model of non-proliferative diabetic retinopathy.</a> Dis Model Mech. 3 (3-4), 236-245 (2010).</li><li>Gleeson, M., Connaughton, V., Arneson, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+hyperglycaemia+in+zebrafish+(Danio+rerio)+leads+to+morphological+changes+in+the+retina.">Induction of hyperglycaemia in zebrafish (Danio rerio) leads to morphological changes in the retina.</a> Acta Diabetol. 44 (3), 157-163 (2007).</li><li>Jorgens, 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=High+tissue+glucose+alters+intersomitic+blood+vessels+in+zebrafish+via+methylglyoxal+targeting+the+VEGF+receptor+signaling+cascade.">High tissue glucose alters intersomitic blood vessels in zebrafish via methylglyoxal targeting the VEGF receptor signaling cascade.</a> Diabetes. 64 (1), 213-225 (2015).</li><li>Lai, A. K., Lo, A. C. <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+diabetic+retinopathy:+summary+and+comparison.">Animal models of diabetic retinopathy: summary and comparison.</a> J Diabetes Res. 2013, 106594 (2013).</li></ol>

作者没有什么可透露的。

缺氧诱导的视网膜血管模型显示, 斑马鱼的分枝点、血管生成芽、总血管面积和系距离均有增加的趋势20。这些发现支持的想法, 斑马鱼是易患糖尿病微血管并发症21, 作为后糖尿病视网膜病变的关键发现包括缺氧介导的血管, 这是强烈的联系 VEGF 表达。高血糖诱导的斑马鱼视网膜的变化导致血管厚度增加, 但保持整体模式22。在葡萄糖溶液中交替浸泡30天也会降低光子和 INL 的厚度23。在斑马鱼中, 葡萄糖代谢物作为血管代谢的支持者的直接影响也已经显示出来。在醛24孵化后, 在斑马鱼胚胎的主干血管中观察到额外的血管 hyperbranching。目前, 没有任何动物模型提供所有的关键标准的糖尿病视网膜病变。早期的变化经常被发现, 但进展到增殖性糖尿病视网膜病变是缺掉的25。斑马鱼也属于这个定义, 因为我们只看到过缺氧介导的血管或高血糖引起的变化, 直到现在。目前的研究结果支持的想法, 斑马鱼是容易受高血糖介导的血管变化, 作为一个动物模型可能显示的进展, 增殖性糖尿病视网膜病变。根据对高血糖介导作用的强度和暴露, 正确的实验模型可能导致斑马鱼视网膜某些区域的缺血, 并促进血管作为增殖性糖尿病视网膜病变的关键标准。然而, 由于斑马鱼在模拟长期微血管病变方面是一个比较新的参与者, 斑马鱼即将推出的糖尿病模型将提供进一步的信息, 并阐明其对其他模型及其病理的重要性。例如, gata1a: DsRed) 斑马鱼与红标红细胞的 tg (fli: EGFP) 线可用于同时可视化潜在的人工 bleedings 在未来斑马鱼模型显示微作为预测进展到民主共和国。
由于视网膜血管进入一连串的拱廊, intervascular 距离、分枝点数和总血管面积的评估与中央视动脉的距离有关。为了避免被评估的血管参数的偏差, 需要一个定位点。IOC 是这样结构和是高度相关的, 因为血管活动区域在直接空间接近度说谎。为了一致的评估, 视网膜扫描应分为多个矩形图像部分与国际奥委会一致的距离。对整个视网膜进行分析, 并对图像进行对称分布。斑马鱼视网膜显示高、低毛细血管密度的区域, 而图像剖面的不均匀分布会导致额外的偏倚。
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/56674/56674fig12.jpg"> 
图 12: 显示血管参数作为视网膜血管可视化的读数的例子.测量内视圆 (IOC) (A)附近的 intervascular 距离 (红色双箭头)。IOC (B)附近有三分支点 (红色圆圈)。斑马鱼视网膜血管的可视化 (红色方框), 显示发芽血管 (C)。从中央动脉 (白色十字) (D)中的一定距离 (白色双箭头) 测量的容器直径。血管密度是由覆盖血管 (对角线红线) (E)占据的视网膜面积的百分比。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56674/56674fig12large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了测量 intervascular 的距离, 你需要设置一定的距离 ioc 作为标准 (图 12A, 白色双箭头) 和画一个假想的水平线 (图 12A, 水平白线) 平行的 ioc。这条线上的船只之间的距离加起来, 算术平均等于 intervascular 距离。每当血管分裂时, 每个图像内都有分枝点, 并从原点继续存在不止一个血管腔。这也包括毛细血管之间的水平连接。重要的是要保持一致的分析, 并决定哪些分支点计数, 并保持这些规则在整个实验, 以减少不必要的变化。血管发芽, 如图 12C所示, 是可以在每个图像中计算的另一个参数, 以评估对视网膜血管的影响。血管生成苗不遵循中央动脉和 IOC 之间的拱廊式的演替, 并聚焦在视网膜的外部部分附近。为了评估某些容器的直径, 始终需要一个定位点, 以确定测量点的距离。中央动脉在视网膜内的来源为主要的茎血管 (图 12D, 白色十字) 提供了这样的指导。被容器占据的区域 (图 12E, 对角线红线) 占整个视网膜面积的百分比是血管密度, 与缺血性区域间接相关。
一个样品的完全共焦扫描应由多个 high-detail 图片组成, 以允许小毛细管 hypersprouting 的可视化。为了优化这一步所花费的时间和资源, 应使用一个自动耕作程序, 并对每个图块进行常规扫描深度。视网膜的不均匀安装可以大大增加用共聚焦显微镜扫描血管的时间。在一个最佳的方法, 你想只扫描血管层 (图 13B, 白盒), 但部分纳入的协鑫通常是必要的。在截断后留下过量的视神经, 以及为达到扁平安装的切口太短, 会导致不均匀的安装。
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/56674/56674fig13.jpg"> 
图 13: 成人斑马鱼视网膜 he 染色和自发荧光的比较.视网膜血管在焦点上面的协鑫(A)。视网膜血管 (白盒) 显示绿色的 EGFP 信号和视网膜层表现出强烈的自发荧光(B)。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56674/56674fig13large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
由于对斑马鱼视网膜血管的正确准备需要事先训练, 小样本大小与未经训练的 dissectors 和强烈变化的准备结果是这项技术的主要限制。虽然制备 tg (fli: EGFP) 斑马鱼的眼睛提供了快速洞察状态的血管, 该技术仍然利用约20分钟的工作时间每视网膜一个有经验的研究员。所有这些视网膜血管的准备步骤必须在解剖显微镜下进行, dissectors 需要一直集中在整个时间, 因为粗心的步骤可能会诱发血管破裂。解剖应定期练习, 因为长期缺席的准备减少了解剖保持船只完整性的可能性。
此外, 通过免疫组化 (IHC) 增加的读数仍然有限, 因为只有少数抗体验证的人和啮齿动物的组织是与斑马鱼工作。对 IHC 感兴趣的实验者建议寻找斑马鱼特有的抗体, 特别是在使用新靶点的时候。另外, 建议使用更多的斑马鱼记者的线, 这是有用的研究细胞以外的眼睛血管。然而, 这一战略是费时的, 因为它需要几个月的时间来生成成人斑马鱼线携带多个转基因记者。
尽管如此, 斑马鱼还是带来了独特的优势。它们相对较小, 容易繁殖。他们可以迅速成长为成人阶段, 他们的胚胎是最佳的药物筛选。这一领域正在迅速发展, 越来越多的文学作品变得容易接近。由于有能力快速生成基因敲除和大量的转基因荧光记者线, 斑马鱼的选择只受选择的研究问题的限制。

糖尿病是一种代谢性疾病, 其定义是由于胰岛素分泌失调或组织对分泌胰岛素的反应不当引起的高血糖。WHO 估计, 在 2014年1 , 4亿2200万成人患有糖尿病, 全球糖尿病患病率预计将增加到 8-10%, 直到 20352, 使糖尿病成为接下来的几十年生活在慢性高血糖导致长期微血管并发症, 包括糖尿病视网膜病变, 肾病, 和神经病。这些并发症的管理和预防是困难的;事实上, 糖尿病正在成为最常见的原因终末期肾病 (末期), 导致透析2, 糖尿病是导致失明的 middle-aged 成人的主要原因3。
糖尿病眼微血管损伤的最初原因是慢性高血糖, 代谢改变, 以及某些危险因素 (如, 高血压, 血脂异常), 导致血管内皮功能障碍, 细胞辍学, 和毛细管回归, 导致脱细胞血管袖。导致视网膜缺血的原因是血管新生和血管通透性增加, 促进了增殖性糖尿病视网膜病变 (民主) 的发展4。糖尿病视网膜病变一般检测到37% 的糖尿病患者, 而视力威胁糖尿病视网膜病变是确定在12% 的筛选白欧洲队列在 UKADS 研究5。目前的治疗只能防止进一步的并发症, 不能完全恢复已经引起的损害。全光凝除血糖控制外, 是增殖性糖尿病视网膜病变的标准治疗方法, 但对相邻的健康组织也有影响。抗 VEGF 干预显示有希望的结果作为替代激光治疗6,7, 但最终, 需要专门的、有针对性的治疗和新药来管理越来越多的有视力丧失风险的病人。
已建立的糖尿病视网膜病变动物研究模型并没有分享人类病理生理学的方方面面。利用正确的物种来解决科研问题的具体要求是实验装置中最重要的部分之一。斑马鱼胚胎 (斑马鱼) 已经被广泛应用于发展研究, 并通过 morpholinos 或 CRISPR/Cas9 技术8, 为击倒或剔除特定基因提供了最佳的实用背景。这些方法可以很容易地用于斑马鱼, 以调查基因, 这是由大规模的基因组范围内的关联研究 (GWAS), 产生洞察到特定机制的疾病进展和敏感性9。短世代时间, 大量的后代, 容易和低成本的处理, 和不断增长的化验支持增加了斑马鱼模型的相关性, 特别是鉴于它的巨大潜力建模代谢疾病。斑马鱼生物机制的保护已被证明是药理治疗发展的基础。例如, 降糖药物二甲双胍和降胆固醇辛伐他汀在 cAMP/地塞米松诱导的高 PEPCK 表达和胆固醇饮食诱发的低胆固醇血症的模型中显示了 "治疗" 的条件10,11,12. 通过实验, 例如: 葡萄糖溶液中的交替培养, 这进一步促进了对总体保守的新陈代谢机制的洞察。链脲佐菌素诱导的β细胞消融, nitroreductase 介导的β细胞消融使用前甲硝唑, 基因糖尿病介导的 pdx1 基因击倒或淘汰赛, 以及增加胰岛素抵抗的模型骨骼肌12. 这些已经建立的协议, 上面提到的物种的具体情况, 并有能力有效地操纵基因组在大量的样本都证明了斑马鱼的优势研究的机制驾驶复杂的疾病过程以及筛选药理干预的能力。
对斑马鱼的基本眼睛解剖 (图 1) 的一般理解是必要的解剖, 以利用斑马鱼作为一个模型的视网膜血管病变。斑马鱼的眼睛有六眼外肌, 四腹直肌, 和两个斜肌, 插入在地球外部的巩膜13。角膜是覆盖晶状体的透明组织, 直接进入巩膜, 形成眼睛的外壳。巩膜是不透明的, 有一个部分光反射表面, 并强烈色素。透镜本身比人的更球形状。视网膜由三个神经元细胞组成, 而提供给氧的视网膜血管与内神经节细胞层紧密相连, 但并不构成视网膜网络。相反, 脉络膜血管位于巩膜和视网膜之间, 与视网膜色素上皮 (RPE) 有关。这个毛细管网络提供氧气对视网膜的外部部分14。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56674/56674fig1.jpg"> 
图 1: 成年斑马鱼眼的示意图描述.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56674/56674fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
利用转基因 tg (fli: EGFP) 斑马鱼线15, 可以直接直观地显示视网膜血管。在血管内皮细胞 fli 启动的控制下表达的绿色荧光蛋白是通过激光扫描显微镜在以后的步骤中进行可视化的基础。这是通过解剖的斑马鱼视网膜和整个安装的组织。这种转基因模型提供了一个快速血管读出没有任何应用血管内标记或全贴装免疫组织化学。为分析斑马鱼中的糖尿病视网膜病变, 每解剖应采用循序渐进的方法和规范化的制剂常规。以下准备协议为其他研究小组提供了选择, 以方便地评估成年斑马鱼眼中血管的变化, 并为斑马鱼视网膜的最佳解剖常规提供指导。

<table><tbody><tr><td>NaOH</td><td>Roth</td><td>6771.3</td><td></td></tr><tr><td>KCl</td><td>Merck</td><td>1.04936</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2*&lt;/sub&gt;6H&lt;sub&gt;2&lt;/sub&gt;0</td><td>Roth</td><td>5239.2</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4*&lt;/sub&gt;7H&lt;sub&gt;2&lt;/sub&gt;0</td><td>Merck</td><td>1.05886.0500</td><td></td></tr><tr><td>Paraformaldehyd</td><td>Roth</td><td>0335.3</td><td></td></tr><tr><td>Sodium dihydrogen phosphate</td><td>Roth</td><td>2370.1</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt (MS-222,Tricaine)</td><td>Sigma</td><td>A5040</td><td></td></tr><tr><td>PBS</td><td>Roth</td><td>9143.1</td><td></td></tr><tr><td>Agarose</td><td>Roth</td><td>2267.3</td><td></td></tr><tr><td>Fluoromount-G</td><td>Thermo-Fischer</td><td>00-4958-02</td><td></td></tr><tr><td>Petri dish</td><td>Greiner Bio one</td><td>633180</td><td></td></tr><tr><td>Six-well plate</td><td>StarLab</td><td>CC7682-7506</td><td></td></tr><tr><td>Needle</td><td>MSG Praxisbedarf</td><td>BD 300900</td><td></td></tr><tr><td>Micro Tweezer</td><td>World Precision Instruments</td><td>14095</td><td></td></tr><tr><td>Microdissection Scissor</td><td>World Precision Instruments</td><td>501778</td><td></td></tr><tr><td>Glass slide</td><td>Carl Roth</td><td>H872.1</td><td></td></tr><tr><td>Coverslip 22mmx22mm</td><td>neoLab</td><td>103512222</td><td></td></tr><tr><td>Scalpel</td><td>MSG Praxisbedarf</td><td>FEA111</td><td></td></tr><tr><td>Epi-Illumination</td><td>Leica</td><td>10446389</td><td></td></tr><tr><td>Fluorescence stereomicroscope MZ10 F</td><td>Leica</td><td>NA</td><td></td></tr><tr><td>Confocal laser-scanning microscope SP5 DS</td><td>Leica</td><td>NA</td><td></td></tr><tr><td>Stereomicroscope M80</td><td>Leica</td><td>NA</td><td></td></tr><tr><td>Zebrafish line: Tg(fli:EGFP), ABTL wildtype</td><td>NA</td><td>NA</td><td>see Reference 15</td></tr><tr><td>Mayer&amp;rsquo;s hematoxylin</td><td>Dr. K. Hollborn &amp;amp; S&amp;ouml;hne</td><td>0088663</td><td></td></tr><tr><td>0.5% eosin</td><td>Dr. K. Hollborn &amp;amp; S&amp;ouml;hne</td><td>NA</td><td></td></tr><tr><td>99,9% ethanol</td><td>Roth</td><td>9065.2</td><td></td></tr><tr><td>Paraffin</td><td>Merck</td><td>1,071,501,000</td><td></td></tr><tr><td>Xylol</td><td>Roth</td><td>4436.2</td><td></td></tr><tr><td>Acetone</td><td>Emsure</td><td>606-001-00-8</td><td></td></tr><tr><td>Microtome RM 2165</td><td>Leica</td><td>NA</td><td></td></tr></tbody></table>

studying diabetes through the eyes of a fish: microdissection, visualization, and analysis of the adult tg(fli:egfp) zebrafish retinal vasculature

糖尿病视网膜病变是 middle-aged 成年人失明的主要原因。全球糖尿病患病率的上升将使糖尿病微血管并发症的预防成为未来几十年的关键研究领域之一。需要专门、有针对性的治疗和新的治疗药物来管理越来越多的视力丧失风险的病人。斑马鱼是一个已建立的动物模型的发展研究问题, 越来越相关的建模代谢多因素疾病的过程。该物种的优势, 允许最佳的可视化和高通量的药物筛选方法, 结合了强大的能力, 敲出感兴趣的基因。在这里, 我们描述一个协议, 将允许简单分析成人 tg (fli: EGFP) 斑马鱼视网膜血管作为一个快速出的设置长期血管病变与血管或血管损害。这是通过解剖的斑马鱼视网膜和整个安装的组织。然后通过在成人视网膜血管中表达的绿 EGFP 记者的共焦显微镜来实现暴露的血管的可视化。正确的组织处理将导致更好的结果和较少的内血管破损, 以确保不变的血管结构的可视化。该方法可用于斑马鱼模型的视网膜血管链接到变化的船舶结构以及血管。

在这里, 我们展示了两个典型的形态学例子的视网膜血管在成人 tg (fli: EGFP) 斑马鱼: 一旦可视化的荧光显微镜 (图 9A) 和一次与共聚焦激光扫描显微镜 (图 9B)。显露的视网膜结构显示高度有组织的样式。在斑马鱼视网膜上, 当用荧光显微镜进行可视化时, 强烈的自发荧光是可见的。因此, 建议只通过共焦扫描的血管层的可视化, 以减少背景荧光和增加对比度。为了获得更快的图像获取或定性数据足够的情况, 荧光显微镜是一种有吸引力的替代品。
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56674/56674fig9.jpg"> 
图 9: 成人 tg (fli: EGFP) 斑马鱼视网膜血管的代表性图片.两个典型的视网膜血管的形态学例子显示: 中央视神经传播到5-7 主要的血管, 然后分支成一连串的拱廊。所有进一步的血管都排入圆周静脉 (CV), 限制每个花瓣的外部部分。通过荧光显微镜在2.5x 放大倍数(A) 中进行可视化。用共聚焦激光扫描显微镜对视网膜血管的可视化通过联合5x5 单图像平铺扫描(B)。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56674/56674fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
光动脉穿透视网膜在视神经头, 并在大多数样本, 传播到5-7 主要血管 (图 10A)。主要的船只, 然后分支到一连串的拱廊, 并连接到内视圈 (IOC), 也称为周向静脉 (CV), 包围在平面安装的视网膜外围的光盘 (见图 10B)。国际奥委会是静脉血管, 引流的动脉血液在眼球球的内缘 (薤)。斑马鱼视网膜血管也显示了高血管活动的区域与毛细血管分支和血管生成萌芽 (图 10C)。这种血管活动主要位于靠近国际奥委会的近距离。重要的是要注意, 同样的眼睛可以显示高和低血管活动在不同地区的视网膜 (比较图 10B和图 10C)。一般而言, 斑马鱼的视网膜血管会显示出更多的缺血性空间, 而在主要分支之间的毛细血管则少于17的小鼠视网膜。每一个样本的眼睛都应该分析, 因为在不同的脉管和单一检查可能导致偏见。
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56674/56674fig10.jpg"> 
图 10: 放大区域成人 tg (fli: EGFP) 斑马鱼视网膜血管可视化.视神经动脉分支到主容器中(A)。视网膜毛细血管在低血管活动区, 连接到图片(B)底部的内视圈 (IOC)。高血管活动区的视网膜毛细血管连接到 IOC (C)。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56674/56674fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
一个高度保守的结构的神经元层已经存在于斑马鱼从大约72小时后受精 (hpf) 后,18。成年斑马鱼视网膜从内而外显示以下几层 (图 11): 神经节细胞层 (协状), 内核层 (光子), 内部细胞核 (INL), 外状层 (组织), 外层核层 (PL), 感光层,和视网膜色素上皮 (RPE)。在图 12中显示了来自视网膜血管可视化的血管参数估计。内视网膜血管位于神经节细胞层 (协鑫) (图 13B, 白盒), 而脉络膜毛细血管则与视网膜色素上皮 (RPE) 有关。
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/56674/56674fig11.jpg"> 
图 11: 成年斑马鱼眼的 he 染色.整个眼睛的横断面概述在透镜的撤除以后(A)。成年斑马鱼眼的视网膜层数19 (B)。PL (C)中自然断开点的指示 (黑色箭头)。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56674/56674fig11large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature at JoVE.com

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

在这里, 我们讨论的方法协议, 将允许简单的分析成人 tg (fli: EGFP) 的斑马鱼视网膜血管作为一个快速出的设置长期血管病理与血管和结构的变化。

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 ...

studying-diabetes-through-eyes-fish-microdissection-visualization

Universidad San Sebastian

Research

JoVE Journal

Medicine

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

13.1K 次观看.  Heidelberg University. 在这里, 我们讨论的方法协议, 将允许简单的分析成人 tg (fli: EGFP) 的斑马鱼视网膜血管作为一个快速出的设置长期血管病理与血管和结构的变化。

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

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

科研

发育生物学

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations 6,7. In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

Trypsin digest is one of the most commonly used methods to analyze retinal vasculature. This manuscript describes the method in detail, including key alterations to optimize the technique and remove the non-vascular tissue while preserving the overall architecture of the vessels.

胰蛋白酶精华协议分析视网膜血管小鼠模型

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

神经科学

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

斑马鱼胚胎眼组织显微切割的

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

生物学

Simultaneous Calcium Imaging and Glucose Stimulation in Living Zebrafish to Investigate In Vivo &#946;-Cell Function

The pancreatic &#946;-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in &#946;-cell function are associated with hyperglycemia that can lead to diabetes. During the process of insulin secretion, &#946;-cells experience an influx of Ca2+. Thus, imaging the glucose-stimulated Ca2+ influx using genetically encoded calcium indicators (GECIs) provides an avenue to studying &#946;-cell function. Previously, studies showed that isolated zebrafish islets expressing GCaMP6s exhibit significant Ca2+ activity upon stimulation with defined glucose concentrations. However, it is paramount to study how &#946;-cells respond to glucose not in isolation, but in their native environment, where they are systemically connected, vascularized, and densely innervated. To this end, the study leveraged the optical transparency of the zebrafish larvae at early stages of development to illuminate &#946;-cell activity in vivo. Here, a detailed protocol for Ca2+ imaging and glucose stimulation to investigate &#946;-cell function in vivo is presented. This technique allows to monitor the coordinated Ca2+ dynamics in &#946;-cells with single-cell resolution. Additionally, this method can be applied to work with any injectable solution such as small molecules or peptides. Altogether, the protocol illustrates the potential of the zebrafish model to investigate islet coordination in vivo and to characterize how environmental and genetic components might affect &#946;-cell function.

Here, the study presents a protocol for calcium imaging and glucose stimulation of the pancreatic &#946;-cells of the zebrafish in vivo.

The pancreatic &#946;-cells sustain systemic glucose homeostasis by producing and secreting insulin according to the blood glucose levels. Defects in ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

多色成像定时转基因斑马鱼：可视化由针对性的神经细胞消融激活视网膜干细胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

从成年斑马鱼中制备新鲜视网膜切片用于前体成像实验

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

成像和胚胎斑马鱼脑血管结构的三维重建

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

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

摘要

探索更多视频

此视频中的章节