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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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>CaCl2*6H20</td>
			<td>Roth</td>
			<td>5239.2</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4*7H20</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&#8217;s hematoxylin</td>
			<td>Dr. K. Hollborn &#38; S&#246;hne</td>
			<td>0088663</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% eosin</td>
			<td>Dr. K. Hollborn &#38; S&#246;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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

13.0K 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

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

定量可视化和皮肤癌症使用动态热成像检测

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

科研

医学

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

斑马鱼模型的糖尿病和代谢记忆

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

技术处理眼睛的视网膜假体植入局部组织病理学分析

With  the recent development of retinal prostheses, it is important to develop  reliable techniques for assessing the safety of these devices in ...

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

为处理眼植入视网膜假体的局部病理组织学分析技术:第2部分视网膜前植入视网膜大头钉

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

瘦素受体<sup&gt;分贝</sup&gt; 2型糖尿病小鼠模型:胰岛分离和活细胞双光子成像完好胰岛

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

通过胰管结扎手术损伤的小鼠胰腺作为模型内分泌和外重新规划和扩散

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析:一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

用于研究小鼠视网膜循环中流动动力学的红细胞和白细胞的荧光染料标记

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...