Name: preparation steps for measurement of reactivity in mouse retinal arterioles ex vivo
Uploaded: 2018-05-08T12:30:00.000+00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo at JoVE.com

这项工作得到了来自 Ernst 和波塔 Grimmke 基金会和德意志 Ophthalmologische 德国 (狗) 的赠款的支持。

本研究的实验程序经德国莱茵兰法动物护理委员会批准。动物护理符合机构指南和视觉和眼科研究协会 (ARVO) 在眼科和视力研究中使用动物的声明。根据欧盟2010/63 号指令 (欧盟) 对动物进行动物实验治疗。男性 C57BL/6J 小鼠 (杰克逊实验室, 酒吧海港, 我, 美国) 3-4 月被用于实验。动物被安置在标准条件下 (温度 23, 2 °c, 湿度范围 55, 10% 和 12 h 光/暗循环), 并获得标准的鼠标周和水ad 随意。
1. 小鼠视网膜的分离
<ol><li>将解剖仪器放在准备表上, 检查所有仪器是否完成, 以便快速准备。</li>
	<li>通过 CO2吸入来牺牲鼠标。</li>
	<li>用钢剪刀斩首鼠标, 用剪刀把头骨 sagitally 切成两半, 用眼剪刀去掉皮肤和大脑 (图 1)。</li>
	<li>用眼剪刀切断颅骨, 只留下眼眶骨。</li>
	<li>将轨道转移到含有冰冷克雷布斯 Henseleit 缓冲液的解剖盘中。</li>
	<li>将解剖盘放在冰上, 或每隔10分钟更换一次缓冲器。</li>
	<li>用眼剪刀切开眼眶骨, 用4型精密镊子和学生 Vannas 弹簧剪刀 (图 2) 轻轻地将眼球地球仪与轨道组织一起移除。</li>
	<li>用5型精密镊子和 Vannas 撕囊术剪刀轻轻去除哈德氏腺体、结缔组织和眼外肌, 注意不要损伤眼动脉的主要分支。</li>
	<li>结扎眼眶分支的眼科动脉和最终的近端的眼科动脉的主要分支使用10-0 尼龙缝合 (图 3)。</li>
	<li>十年代使用4型精密镊子将眼球转移到含有70% 乙醇的培养皿中。将眼球放回解剖盘中, 用新鲜的克雷布斯溶液替换缓冲液。在乙醇暴露后, 角膜会呈白色。</li>
	<li>用30口径的针刺穿周围的角膜, 用 Vannas 撕囊术剪刀解剖角膜。轻轻切下虹膜组织, 在脉络膜和视网膜之间放置一 Vannas 撕囊术剪刀的尖点, 并切断脉络膜和巩膜。在视神经周围留一些巩膜组织 (图 4)。</li>
</ol>2. 在灌注腔内安装视网膜
注: 用于视网膜动脉实验的灌注腔是自制的。它由一个透明的储层组成, 里面有流出管 (图 5)。硅胶管的一端用 histoacryl 粘合剂粘在房间的底部, 另一端附着在三路旋塞阀上。将含有新鲜克雷布斯缓冲器的注射器连接到三路旋塞阀, 然后用缓冲器填满管子。
<ol><li>拿一个玻璃微与针持有人, 并将其推入管的末端, 在灌注室的底部。然后用4型剪刀打破微的尖端, 获得100µm 直径的尖端。</li>
	<li>将焦点放在微的尖端上, 然后将其刷新通过与管连接的注射器。注意毛细血管不被碎片遮挡。如果堵塞, 取出, 冲洗油管和插入另一个微。</li>
	<li>用冷克雷布斯缓冲液填充灌注腔, 并将腔室置于立体显微镜下。</li>
	<li>将透明的塑料平台放进灌注室。该平台有一个1.8 毫米的压痕宽度为视神经和眼科动脉, 并放置在两条直径为1.6 毫米的钢丝上。然后, 在平台上放置一个2.8 毫米内径和4.0 毫米外径的不锈钢环。</li>
	<li>用勺子将视网膜与视神经转移, 并将眼部动脉从解剖室连接到灌注室。这一步骤很重要, 以防止潜在的伸展组织, 因为它可能是有害的实验。</li>
	<li>切除眼动脉近端的缝合部分, 将两个预制的10-0 尼龙缝合环放到微上, cannulate 眼动脉与微。将动脉栓在微 (图 6) 上, 并使用细点镊子将视网膜置于透明平台上。</li>
	<li>用两个细点镊子轻轻撕开前透镜胶囊, 拔出透镜, 用 microscissors 切断整个透镜胶囊。</li>
	<li>随后, 使四径向切口进入视网膜一半距离从扁平到视神经, 并放置一个不锈钢环到视网膜上修复它的底部。视网膜现在已经准备好进行实验 (图 7)。</li>
</ol>3. 实验用视网膜微动脉的制备
<ol><li>在光镜下放置灌注室。目的是一个100X 水浸泡物镜。</li>
	<li>将两个油管连接进进出流至周环泵, 并将克雷布斯缓冲氧合的燃烧室与 95% O2和碳酸与 5% CO2在37摄氏度之间循环。使用100至120毫升/分钟之间的流速。</li>
	<li>将与微连接的硅胶管连接到储层硅胶软管通过三路旋塞阀。然后, 用克雷布斯缓冲器将储层填入与50毫米汞对应的高度, 但不要打开三路旋塞阀。</li>
	<li>仔细观察显微镜的目标, 聚焦于视网膜表面。寻找血管或红细胞。血管可能完全塌陷, 所以有时只有一些红细胞可见。</li>
	<li>当发现血管时, 关注它, 打开三路旋塞阀。该血管的直径应立即增加, 红血球移动要么离心, 如果它是一个动脉, 或向心地的情况下静脉。与同一顺序的静脉相比, 小动脉直径较小。</li>
	<li>一旦发现了具有良好可见壁的血管, 就可以在30分钟的平衡周期 (图 8) 下进行实验。在平衡期内, 小动脉只发育微弱的内在音调, 导致腔内直径减少10%。</li>
</ol>4. 执行实验
<ol><li>通过将血栓模拟 911-双脱氧-9α、11α-methanoepoxy 前列腺素 F 2α (U46619), 通过将其添加到浴溶液中, 测试容器的生存能力. 该剂收缩鼠视网膜小动脉约 50%, 从静止直径的浓度≥ 10-6 m。</li>
	<li>一旦该船只被确认可行, 通过循环泵, 并提供新鲜的克雷布斯缓冲液在浴缸中清洗激动剂。</li>
</ol>

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作者没有竞争的金融利益。

由于视网膜血管的体积较小, 对小鼠视网膜血管反应的测量具有挑战性。采用透射光镜对视网膜微动脉进行了可视化处理。这是可能的, 因为孤立的视网膜是半透明的。该技术的优点是高光学分辨率。计算的空间分辨率为11像素/µm。然而, 这个使用白光的光学系统的真正分辨率介于200和 300 nm 之间, 这是由阿贝衍射极限所解释的。由于小鼠视网膜动脉的第一个分支的内径为20至30µm, 因此该系统可检测到约1% 的直径变化。在其他研究报告中, 不需要额外的技术设备或荧光染料来可视化小的视网膜微动脉5,6,7。技术的缺点是长的准备时间, 是在90到120分钟之间由被训练的调查员。如果制备时间超过180分钟, 内皮功能开始衰减。
这项技术有几个重要的关键步骤。首先, 彻底结扎所有球动脉分支是很重要的。如果结扎不全, 视网膜动脉不会因渗漏而加压。第二, 在打开眼球之前, 在十年代的乙醇浸泡是很重要的, 以解除眼动脉平滑肌的反应。我们以前证明, 浸泡十年代在70% 乙醇完全停用眼部动脉平滑肌8。相比之下, 视网膜小动脉不太可能直接受到乙醇的影响, 因为它们受到球壁的保护。如果省略此步骤, 眼科动脉的直径变化可能会影响视网膜的灌注。值得注意的是, 眼科动脉的反应性可能与视网膜微动脉的反应有明显差异, 以回应同一激动剂9,10。第三, 避免眼动脉扭转。特别是在将视网膜准备安装到平台上时, 眼动脉可能会扭曲, 导致腔闭塞。此外, 仔细检查, 玻璃毛细管的尖端没有闭塞, 以使加压的视网膜小动脉。我们建议不要在同一视网膜准备中连续执行超过三个浓度-反应曲线, 因为我们观察到, 当重复浓度-反应曲线超过三次时, 乙酰胆碱的反应变得较弱。然而, 根据所应用的药剂, 浓度响应曲线的重复性可能存在差异, 因此应单独进行测试。
我们在研究中应用了药理学药物 extraluminally, 尽管使用伺服控制泵进行腔内灌注也是可能的。由于该技术允许测量小实验动物, 包括小鼠和大鼠视网膜血管反应性的局部机制, 利用转基因动物的基因靶向技术的优势可以用这种方法来获取.

视网膜灌注紊乱涉及各种眼部疾病的发病机制, 如糖尿病视网膜病变、高血压视网膜病变和青光眼1,2,3。因此, 旨在测量视网膜血管反应性的研究对于了解这些疾病的病理生理学和制定新的治疗方法很重要。
由于基因操作在小鼠基因组中的可能性, 老鼠已成为一个广泛使用的动物模型的研究心血管系统的4。然而, 由于视网膜血管体积小 (≤30µm), 对小鼠视网膜血管反应性的测量具有挑战性。例如, 用于体内测量的 stereomicroscopic 技术在其光学分辨率上受到限制, 因此仅允许精确检测小于≤30µm 直径的小血液中直径或血流量的变化, 同时配备其他尖端设备, 如使用荧光染料的共焦显微镜或自适应光学扫描光检眼镜 5, 6.此外, 对体内测量的解释, 旨在确定视网膜血管内的局部信号机制, 可以混淆麻醉, 系统血压的变化和球血管的影响。
因此, 我们开发了一种方法来测量小鼠视网膜血管的反应, 高光分辨率的前体。本文提出的技术允许可视化视网膜微动脉通过透射光显微术。该方法也可用于大鼠眼血管研究中, 为基因靶向技术的优势提供了途径。

<table><tbody><tr><td>Steel Scissors</td><td>Carl Roth GmbH</td><td>3576.1</td><td>1x 140 mm</td></tr><tr><td>Eye Scissors</td><td>Geuder</td><td>G-19390</td><td>1x straight, 10.5 cm</td></tr><tr><td>Precision tweezers, straight with fine tips</td><td>Carl Roth GmbH</td><td>LH68.1</td><td>2x type 4</td></tr><tr><td>Precision tweezers, straight with extra fine tips</td><td>Carl Roth GmbH</td><td>LH53.1</td><td>2x type 5</td></tr><tr><td>Vannas capsulotomy scissors</td><td>Geuder</td><td>19760</td><td>1x straight, 77 mm</td></tr><tr><td>Student Vannas Spring Scissors</td><td>Fine Science Tools</td><td>91501-09</td><td>1x curved,</td></tr><tr><td>&lt;em&gt;Barraquer&lt;/em&gt; Needle Holder</td><td>Geuder</td><td>G-17500</td><td>1x curved, 120 mm</td></tr><tr><td>Needle</td><td>Becton, Dickinson and Company</td><td>305128</td><td>1x 30 G</td></tr><tr><td>Glass Capillaries (for producing micropipettes)</td><td>Drummond Scientific Company</td><td>9-000-1211</td><td>1x (1.2 x 0.8 mm; outer/inner diameter)</td></tr><tr><td>Nylon Suture</td><td>Alcon</td><td>198001</td><td>1x 10-0</td></tr><tr><td>Nunclon cell culture dish</td><td>Thermo Fisher Scientific</td><td>153066</td><td>1x 35 mm diameter</td></tr><tr><td>Nunclon cell culture dish</td><td>Thermo Fisher Scientific</td><td>172931</td><td>1x 100 mm diameter</td></tr><tr><td>Discofix C</td><td>Braun</td><td>16500C</td><td>10 cm</td></tr><tr><td>Histoacryl adhesive</td><td>B. Braun Surgical, S.A.</td><td>1050052</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pericyclic pump&amp;nbsp; (CYCLO II)</td><td>Carl Roth GmbH</td><td>EP76.1</td><td>1x</td></tr><tr><td>Vertical Pipette Puller Model 700C</td><td>David Kopf Instruments</td><td></td><td>1x</td></tr><tr><td>Microscope (Vanox-T AH-2)</td><td>Olympus</td><td></td><td>1x</td></tr><tr><td>Water immersion objective LUMPlanFL, 1.0 NA</td><td>Olympus</td><td></td><td>1x</td></tr><tr><td>Digital camera (TK-C1381)</td><td>JVC</td><td></td><td>1x</td></tr><tr><td>Perfusion chamber</td><td>self-made</td><td></td><td>1x</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Drugs and Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ethanol</td><td>Carl Roth GmbH</td><td>K928.4</td><td></td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>5239.1</td><td></td></tr><tr><td>Kalium chloride (KCl)</td><td>Carl Roth GmbH</td><td>6781.1</td><td></td></tr><tr><td>Kalium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>3904.2</td><td></td></tr><tr><td>Magnesium sulphate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>261.2</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Carl Roth GmbH</td><td>9265.2</td><td></td></tr><tr><td>Sodium hydrogen carbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>0965.3</td><td></td></tr><tr><td>&amp;alpha;-(D)-(+)- Glucose monohydrate</td><td>Carl Roth GmbH</td><td>6780.1</td><td></td></tr><tr><td>9,11-dideoxy-9&amp;alpha;,11&amp;alpha;-methanoepoxy prostaglandin F2&amp;alpha; (U-46619)</td><td>Cayman Chemical</td><td>16450</td><td></td></tr><tr><td>Acetylcholine chloride</td><td>Sigma-Aldrich</td><td>A6625-25G</td><td></td></tr></tbody></table>

preparation steps for measurement of reactivity in mouse retinal arterioles ex vivo

血管功能不全和正常视网膜灌注的改变是各种视危眼部疾病发病的主要因素, 如糖尿病视网膜病变、高血压视网膜病变、可能青光眼等。因此, 视网膜微血管制剂是生理学和药理学研究的关键工具, 它描绘了潜在的病理生理机制, 并设计了治疗疾病的方法。尽管小鼠模型在眼科研究中的广泛应用, 但对视网膜血管反应性的研究在这个物种中是稀缺的。这一差异的一个主要原因是, 由于这些视网膜血管的体积小, µm 的隔离程序是有挑战性的, 这是30的腔内直径。为了规避这些视网膜微血管直接分离功能研究的问题, 我们建立了一种隔离和制备技术, 使体外在近生理条件下对小鼠视网膜 vasoactivity 的研究.虽然目前的实验准备将具体地提到老鼠视网膜小动脉, 这种方法可以很容易地用于微血管从大鼠。

U-46619 在 C57Bl/6J 背景的野鼠视网膜小动脉中产生浓度依赖性收缩反应。在浓度为 10-6 M 时, 管腔直径的减少≈50% 于静息直径。图 9A显示一个视网膜动脉的代表性浓度-反应曲线。在 U46619 的微动脉 preconstricted 中, 乙酰胆碱诱发浓度依赖性增加的管腔直径≈25% 从 preconstricted 直径在 10-5 M, 指示一个完整的血管内皮 (图 9B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56199/56199fig1.jpg"> 图 1: 对鼠标头骨的解剖.被斩首的老鼠头上的皮肤被移除以暴露眼睛和眼眶。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56199/56199fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56199/56199fig2.jpg"> 图 2: 眼睛地球仪和眼眶组织的隔离.眼眶骨用眼剪刀切开, 眼球与球组织和视神经密切隔绝。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56199/56199fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56199/56199fig3.jpg"> 图 3: 眼睛地球仪和眼科动脉的制备.一旦周围的眼眶组织解剖细 microscissors, 眼动脉被仔细暴露, 他们的小分支结扎10-0 尼龙单丝缝合。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56199/56199fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56199/56199fig4.jpg"> 图 4: 眼部结构的解剖.为了形象化视网膜, 巩膜和 uvea 用 Vannas 撕囊术剪刀解剖。一些巩膜组织周围的视神经被留下来避免损伤的眼科动脉。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56199/56199fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56199/56199fig5.jpg"> 图 5: 用于实时视频显微术的风琴室.房间是自制的。它包括一个100毫米直径的培养皿与进出流管。这些管子用 histoacryl 粘合剂粘在一起。外充氧和碳酸克雷布斯-Henseleit 缓冲由蠕动泵通过这些管传播。硅胶管的一端粘在房间的底部, 另一端附着在三路旋塞阀上。在管子的末端, 一个玻璃微与100µm 的尖端插入了, 为眼科动脉的插管服务。通过硅胶管, 通过将克雷布斯缓冲器的硅胶管填充到与50毫米汞对应的水平, 使眼部循环加压.<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56199/56199fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56199/56199fig6.jpg"> 图 6: 眼科动脉插管.眼科动脉空心与玻璃微和缝合10-0 尼龙缝合。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56199/56199fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56199/56199fig7.jpg"> 图 7: 眼科动脉插管.将视网膜放到透明的平台上后, 取出囊袋的透镜, 将四个径向切口制成视网膜, 并将2.8 毫米内径和4.0 毫米外径的不锈钢环放到视网膜上, 将其固定到 b 上。ottom。然后视网膜就准备好进行实验了。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56199/56199fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56199/56199fig8.jpg"> 图 8: 视网膜血管的可视化.一种典型的视网膜动脉, 里面有红血球。
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56199/56199fig9.jpg"> 图 9: 使用视频显微镜的功能研究.单一视网膜动脉的代表性浓度反应曲线。(A) 收缩、U46619 (10-9至 10-6 M) 的浓度响应曲线, (B) 用于内皮依赖性血管扩张剂、乙酰胆碱 (10-8至 10-5 m)。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56199/56199fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo at JoVE.com

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

许多视力威胁的眼部疾病与功能失调的视网膜微血管有关。因此, 测量视网膜动脉的反应是重要的, 以调查潜在的病理生理学机制。本文描述了一个详细的协议, 为小鼠视网膜动脉隔离和准备, 以评估血管活性物质的作用, 以对维管径。

小鼠视网膜微动脉的反应性测定的制备步骤前体

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

preparation-steps-for-measurement-reactivity-mouse-retinal-arterioles

Universidad San Sebastian

Research

JoVE Journal

Medicine

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

7.1K 次观看.  University Medical Center of the Johannes Gutenberg University Mainz. 许多视力威胁的眼部疾病与功能失调的视网膜微血管有关。因此, 测量视网膜动脉的反应是重要的, 以调查潜在的病理生理学机制。本文描述了一个详细的协议, 为小鼠视网膜动脉隔离和准备, 以评估血管活性物质的作用, 以对维管径。

视频: Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

科研

神经科学

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

医学

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

非增殖性和增殖性视网膜病变小鼠整个视网膜血管参数的定量

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

体内 小鼠视网膜血管损伤读数以促进可重复性

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

从小鼠荧光血管造影图像中测量视网膜血管直径

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

体外细胞生理学研究中视网膜微动脉的分离

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

生物学

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

细胞外通量分析技术在视网膜组织外种中的能量代谢测定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

小鼠视网膜微动脉的反应性测定的制备步骤前体

摘要

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此视频中的章节

血管再生在中枢神经系统中使用的小鼠视网膜评估

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

非增殖性和增殖性视网膜病变小鼠整个视网膜血管参数的定量

体内小鼠视网膜血管损伤读数以促进可重复性

从小鼠荧光血管造影图像中测量视网膜血管直径

体外细胞生理学研究中视网膜微动脉的分离

细胞外通量分析技术在视网膜组织外种中的能量代谢测定

小鼠视网膜微动脉的反应性测定的制备步骤前体

摘要

探索更多视频

此视频中的章节

血管再生在中枢神经系统中使用的小鼠视网膜评估

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

罗登斯缺血性视网膜疾病的氧诱导视网膜病变模型

非增殖性和增殖性视网膜病变小鼠整个视网膜血管参数的定量

体内 小鼠视网膜血管损伤读数以促进可重复性

从小鼠荧光血管造影图像中测量视网膜血管直径

体外细胞生理学研究中视网膜微动脉的分离

细胞外通量分析技术在视网膜组织外种中的能量代谢测定

体内小鼠视网膜血管损伤读数以促进可重复性