Name: preparation steps for measurement of reactivity in mouse retinal arterioles ex vivo
Uploaded: 2018-05-08T12:30:00
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。
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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...

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>Barraquer 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pericyclic pump&#160; (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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Drugs and Solutions</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 (CaCl2)</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 (KH2PO4)</td>
			<td>Carl Roth GmbH</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulphate (MgSO4)</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 (NaHCO3)</td>
			<td>Carl Roth GmbH</td>
			<td>0965.3</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-(D)-(+)- Glucose monohydrate</td>
			<td>Carl Roth GmbH</td>
			<td>6780.1</td>
			<td></td>
		</tr>
		<tr>
			<td>9,11-dideoxy-9&#945;,11&#945;-methanoepoxy prostaglandin F2&#945; (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</stro...

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。

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