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>

<ol>
	<li>Toda, N., Nakanishi-Toda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide:+ocular+blood+flow,+glaucoma,+and+diabetic+retinopathy.">Nitric oxide: ocular blood flow, glaucoma, and diabetic retinopathy.</a> Prog Retin Eye Res. 26 (3), 205-238 (2007).</li><li>Schuster, A. K., Fischer, J. E., Vossmerbaeumer, C., Vossmerbaeumer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography-based+retinal+vessel+analysis+for+the+evaluation+of+hypertensive+vasculopathy.">Optical coherence tomography-based retinal vessel analysis for the evaluation of hypertensive vasculopathy.</a> Acta Ophthalmol. 93 (2), e148-e153 (2015).</li><li>Cherecheanu, A. P., Garhofer, G., Schmidl, D., Werkmeister, R., Schmetterer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+perfusion+pressure+and+ocular+blood+flow+in+glaucoma.">Ocular perfusion pressure and ocular blood flow in glaucoma.</a> Curr Opin Pharmacol. 13 (1), 36-42 (2013).</li><li>Faraci, F. M., Sigmund, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+biology+in+genetically+altered+mice+:+smaller+vessels,+bigger+insight.">Vascular biology in genetically altered mice : smaller vessels, bigger insight.</a> Circ Res. 85 (12), 1214-1225 (1999).</li><li>Kornfield, T. E., Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Retinal+Blood+Flow+Using+Fluorescently+Labeled+Red+Blood+Cells.">Measurement of Retinal Blood Flow Using Fluorescently Labeled Red Blood Cells.</a> eNeuro. 2 (2), (2015).</li><li>Guevara-Torres, A., Joseph, A., Schallek, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label+free+measurement+of+retinal+blood+cell+flux,+velocity,+hematocrit+and+capillary+width+in+the+living+mouse+eye.">Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye.</a> Biomed Opt Express. 7 (10), 4229-4249 (2016).</li><li>Schallek, J., Geng, Y., Nguyen, H., Williams, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+topography+of+retinal+pericytes+in+the+living+mouse+retina+using+in+vivo+adaptive+optics+imaging+and+ex+vivo+characterization.">Morphology and topography of retinal pericytes in the living mouse retina using in vivo adaptive optics imaging and ex vivo characterization.</a> Invest Ophthalmol Vis Sci. 54 (13), 8237-8250 (2013).</li><li>Gericke, 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=Identification+of+the+muscarinic+acetylcholine+receptor+subtype+mediating+cholinergic+vasodilation+in+murine+retinal+arterioles.">Identification of the muscarinic acetylcholine receptor subtype mediating cholinergic vasodilation in murine retinal arterioles.</a> Invest Ophthalmol Vis Sci. 52 (10), 7479-7484 (2011).</li><li>Gericke, 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=Functional+role+of+alpha1-adrenoceptor+subtypes+in+murine+ophthalmic+arteries.">Functional role of alpha1-adrenoceptor subtypes in murine ophthalmic arteries.</a> Invest Ophthalmol Vis Sci. 52 (7), 4795-4799 (2011).</li><li>Bohmer, 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=The+alpha(1)B+-adrenoceptor+subtype+mediates+adrenergic+vasoconstriction+in+mouse+retinal+arterioles+with+damaged+endothelium.">The alpha(1)B -adrenoceptor subtype mediates adrenergic vasoconstriction in mouse retinal arterioles with damaged endothelium.</a> Br J Pharmacol. 171 (16), 3858-3867 (2014).</li></ol>

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...

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。

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

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

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

前溶瘤病毒的活组织的体内感染

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

科研

医学

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

在体测量小鼠肺血管内皮表面层

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

视网膜胶质细胞活化的体内动力学在神经退行性疾病:共聚焦成像眼底及细胞形态计量小鼠青光眼

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

小鼠右心室舒张参数的超声心电图测量

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

增殖性糖尿病视网膜病变纤维血管并发症的体外组织培养模型

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

古树康 (GSK) 颗粒的制备,用于 Vivo 和体外实验

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

小鼠视网膜微动脉的反应性测定的制备步骤前体

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前溶瘤病毒的活组织的体内感染

在体测量小鼠肺血管内皮表面层

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

在体内 ^19女性 MRI对细胞跟踪

一个

视网膜胶质细胞活化的体内动力学在神经退行性疾病:共聚焦成像眼底及细胞形态计量小鼠青光眼

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

小鼠右心室舒张参数的超声心电图测量

增殖性糖尿病视网膜病变纤维血管并发症的体外组织培养模型

古树康 (GSK) 颗粒的制备,用于 Vivo 和体外实验