The authors wish to thank Drs. David Calkins (Vanderbilt University) and James Morgan (Cardiff University) for sharing their expertise and for helpful advice towards developing this procedure. This study was supported by grants from the Canadian Institutes of Health Research (A.D.P.). Y.A.I. and N.B. are the recipients of postdoctoral fellowships from the Fonds de recherche du Qu&#233;bec-Sant&#233; (FRQS). N.B. was awarded a H.H. Jasper scholarship from the Groupe de Recherche sur le Syst&#232;me Nerveux Central (GRSNC). A.D.P. is a Chercheur Boursier National FRQS.

下面的程序是符合加拿大议会关于动物饲养对实验动物的使用以及在视觉与眼科（ARVO）眼科使用动物及视觉研究从研究协会声明的指导方针进行。 1.微针的制备前房内注射<ol><li>带拉出器，生成从硼硅玻璃毛细管的微针</li><li>在显微镜下，用锋利的刀片，以精心打造的微针尖端的开口。所得开口应具有约190微米和70微米的长轴和短轴直径为椭圆形，分别。测量所制备的微针开口由第一获取图像与显微镜利用图像分析软件，随后通过定量下置于一个标尺的面积。 </li><li>在微量斜边系统，将微针在20 degre相对角度E要斜切板，使微针开口接触到板。斜角为大约10分钟，直到边缘是平的，光滑的。加蒸馏水几滴在过程中提供帮助。 </li><li>旋转微针斜角围绕开口，直到提示是尖锐的两条边。 </li><li>清洁所有碎片和水使用喷雾剂掸子微针末端开口。 </li><li>仔细检查在显微镜下完成的微针。骨折丢弃微针在手术过程中，以尽量减少微针断裂的风险。 </li><li>用乙醇漂洗，然后用无菌平衡盐溶液（BSS）消毒微针。 </li></ol> 2.磁性微珠溶液的制备注意:在此研究中使用的磁微珠涂覆有环氧基。为了防止任何不利的影响，如珠粒结块和不需要的分子间的相互作用，这些环氧基必须首先从微珠与注射手术操作前除去。 <ol><li> 从磁珠环氧基团的去除 <ol><li>制备的0.02M氢氧化钠（NaOH，MW39.997克/摩尔）在10倍的Tris缓冲液中的溶液（MW121.14克/摩尔）。 </li><li>轻轻涡旋磁微珠溶液的股票（直径4.5微米，4×10 8珠/ ml）的，直到珠粒均匀悬浮在溶液中。 </li><li>迅速吸取1毫升磁珠溶液到50ml的0.02M NaOH的10×Tris缓冲液。 </li><li>旋转在RT 24小时，从珠除去环氧基。 </li><li>用磁铁固定到管的底部收集珠。水平定向管，以确保所有的珠被吸引到磁铁。旋转在室温额外的4小时。 </li><li>随着微管，小心地取出上清而不干扰珠沉淀。 </li><li>轻轻涡旋沉淀在50毫升10X的TRIS缓冲区，直到珠以及暂停。 </li><li>重复步骤2.1.4至2.1.6。 </li></ol></li><li> 浓度和在无菌平衡盐溶液磁微珠的重悬 </li></ol>注意:有必要集中在无菌平衡盐溶液（BSS）磁珠溶液的库存达到1.6×10 6珠/微升的最终浓度，使2.4×10 6珠可在注射入前房1.5微升，这是适合于小鼠眼的最终体积。 <ol><li>通过2分钟轻轻涡旋洗珠在5ml超纯水实验室级水。 </li><li>通过吸引他们与磁铁的管底部收集珠。 </li><li>随着微管，小心地取出水，无干扰的珠沉淀。 </li><li>重复步骤2.2.1至2.2.3三次。 </li><li>在层流罩，洗珠用500μlBSS由pipettin克上下。执行在层流罩无菌条件下，本节剩下的步骤。 </li><li>通过吸引他们与磁铁的管底部收集珠。 </li><li>随着微管，小心地取出BSS不干扰珠子的沉淀。 </li><li>重复步骤2.2.5至2.2.7三次。 </li><li>通过上，在250微升BSS的上下吹吸珠悬浮。 </li><li>确保该珠子溶液是良好均化。然后，迅速分装25微升悬浮液进入无菌0.5毫升管。股票珠溶液的最终浓度为1.6×10 6珠/微升。 </li><li>保存在4℃。 </li></ol> 3.高眼压的诱导注:第3节是两个人的操作。在一个特定的操作是由一个特定的人执行的情况下，适当的人被识别。一般情况下，1人负责处理在显微镜下鼠标，而人2为responsible用于操纵微量注射泵。手术过程的总持续时间应少于10分钟（步骤3.9至3.17）。 <ol><li>在成年C57BL / 6小鼠进行的程序。家鼠与获得食物和水随意的标准环境。在本文中，使用3和4.5个月的年龄之间的雌性C57BL / 6小鼠。然而，该协议可以适应男性和不同年龄的小鼠，以及其它小鼠品系，包括转基因和基因敲除小鼠。 </li><li>使用校准反弹眼压计测量麻醉和微珠注射前清醒小鼠基线眼压。应用角膜盐酸丙美卡因一滴。 <ol><li>按住两耳之间的皮肤轻轻地抑制鼠标。放置在台式鼠标，使得动物的舒适和眼睛是可访问的。持张力计垂直于角膜表面，并采取至少三组连续10次读数每眼，得到眼压平均水平。眼压清醒小鼠的测量最好绕过IOP麻醉相关的影响。可替代地，眼压可以在anesthetised小鼠步骤3.4之后测量。 </li></ol></li><li>制备的20毫克/毫升氯胺酮，2毫克/毫升赛拉嗪，和0.4毫克/毫升乙酰丙嗪组成的库存鼠标鸡尾酒麻醉混合物。 </li><li>由鸡尾酒混合物（1微升/ g体重的）的腹腔内给药诱发小鼠麻醉。利用可注射的麻醉剂鸡尾酒优于气体麻醉剂（ 例如，异氟烷），因为它允许灵活处理鼠标头时作为动物是未连接到吸入面罩。此外，可注射的麻醉剂需要较长的恢复期确保微珠在虹膜角膜角沉降而不撞出回进入前房。 </li><li>辖0.05毫克每公斤体重丁丙诺啡皮下公斤。 </li><li>治疗眼睛有托EYË下降诱发瞳孔扩大。由于鼠前房的小尺寸，瞳孔必须散瞳在注射过程中很容易地显现微针的定位和进步。 </li><li>在对侧眼（未操作）适用外用药膏，以避免在操作过程中的角膜的干燥。 </li><li>附加一个干净的微针的微量注射泵的喷射组件。每次操作后，更换微针，避免交叉污染的动物。 </li><li>人1:anesthetised鼠标传送到操作平台。在显微镜下，确保瞳孔充分散瞳和眼部肌肉放松，这样就没有眼球运动。没有眼睛运动的注射过程中确保稳定。轻轻擦拭使用吸水擦拭眼周托吡卡胺滴眼液。 </li><li>人2:通过上下吹打混匀磁微珠溶液。 </li><li>使用微量注射泵，立即加载MICRoneedle 1.5微升均质磁微珠溶液（2.4×10 6珠）的（在第1制备）。确保气泡在微针的尖端不存在。微针被加载后，开展尽快步骤3.12 3.13使磁性微珠溶液保持在均匀的悬浮液。 </li><li>在45°角的装微针，前方相对放置在角膜缘定位。人1:支持使用塑料镊子的眼睛。确保微针和塑料镊子之间的角度是大约90°。 </li><li>人2:随着装微针，使微针的尖端进入前房轻轻穿刺角膜。确保装入微针保持在相对于45°角的角膜缘穿刺期间。避免镜头或虹膜任何接触。确保微针不进入后房。人1:继续支持眼睛使用塑料镊子。 </li><li>人1:无移动鼠标头，放置磁铁眼睛旁边，相对的微针尖端，以吸引磁珠进入前房，并尽量减少与角膜的内表面的珠接触。人2:使用微量注射泵，注射1.5微升磁珠溶液进入前房。微珠溶液注入历时15至30秒的。人1:继续注射的整个持续时间期间保持所述磁体相对的微针尖端。 </li><li>人2:一旦珠的全部体积已经注入，慢慢撤出从眼睛的微针。人1:为了避免微珠回流，继续保持旁边的眼磁铁另外30至60秒，以吸引朝向前房磁珠。 </li><li>人1:使用磁铁，吸引珠的虹膜角膜角。确保珠形成一个均匀分布的环围绕前房的周长。在此步骤中，避免吸引小珠的角膜，因为他们必须坚持在接触角膜的内表面上的倾向。 </li><li>治疗所操作的眼用抗生素滴眼剂以减少感染的风险。 </li><li>让鼠标在隔热垫恢复，直到完全清醒（〜3至4小时）。将鼠标，使得操作眼朝上。这种定位将防止注入的珠粒积累由引力角膜的内表面上。此外，该位置将减少感染的操作眼将不会与任何被褥和/或可以存在于所述笼的其它材料接触的可能性。在动物遇险显示任何征兆的情况下，需要额外的管理剂量丁丙诺啡。 </li><li>允许小鼠从程序取眼压测量之前恢复至少2天。在3.2中所述测量眼压。莫尼器眼压至少每周一次或更频繁，如需要的话，并在一天的同一时间，以减少昼夜相关的波动。 </li><li>眼压测量，使用对侧眼从操作鼠标作为内部对照。另外，使用来自完好，无操作或假手术小鼠为幼稚控制测量。 </li></ol> 4.视网膜评估神经节细胞索玛和轴突生存<ol><li>灌注通过的0.1M磷酸盐缓冲液（PBS）然后立即冰冷的4％多聚甲醛（PFA）心脏内注射进行眼压的小鼠。 </li><li>灌注完好，非手术小鼠为4.1，并且使用它们作为未受伤的控制。不推荐使用从手术小鼠对侧眼睛，因为受伤后的变化对侧眼睛已经报15,16，可以混淆数据解释，以评估RGC存活。另外，使用假手术的眼睛注射10.5微升的BSS作为对照。 </li><li>使用microscissors，精心裁剪结缔组织眼部周围，将其从眼窝隔离。通过在视神经乳头的水平切割其分离从眼视神经。 </li><li>使用地下30针，使一个孔中的角膜，以允许固定剂溶液到眼睛中的渗透。将眼睛在4％PFA，在4℃孵育1小时额外的固定。 </li><li>放置视神经在含有2％的PFA和在0.1M二甲胂酸钠2.5％戊二醛溶液（MW 214克/摩尔）和孵育O / N，在4℃再固定。 </li></ol> 5. RGC索玛密度对量化平置式视网膜 注意 :以下过程是由纳达尔，尼古拉斯等[17]的协议适配，并概述使用对脑特异性同源盒/ POU域蛋白3A（Brn3a）对视网膜平板支架的抗体视网膜神经节细胞的量化。替代甲基用于标记的RGC ODS也可以使用包括与针对具有多个剪接（RBPMS）或逆行标记用荧光金或的DiI RNA结合蛋白的抗体免疫组织化学。 <ol><li>在解剖显微镜下，用直至角膜分离容易制作切口沿着整个角膜缘移除眼睛的前部。取下角膜和晶状体。通过使沿锯齿缘和视神经削减仔细分离从眼睛的视网膜上。 </li><li>通过对视神经清楚地界定这四个象限的视网膜对视网膜的边缘使四个小切口等距准备视网膜平山。用小刷子，轻轻地从视网膜删除任何剩余的玻璃体。尽可能去除尽可能多的玻璃体是获得清洁力强的免疫组织化学信号的关键。 </li><li>仔细视网膜传送到含有0.5％的Triton X-100的PBS 48孔平底培养板中，以使视网膜是自由浮动。确保神经节细胞层朝上。 </li><li>放置在-70℃下将培养板15分钟。在PBS中洗涤解冻视网膜，进一步通透两次在新鲜的2％的Triton X-100之后。 </li><li>稀释Brn3a抗体0.3 - 0.5微克/毫升含2％的Triton X-100和2％正常驴血清的PBS。通过在4℃下轻轻摇动O / N孵育在Brn3a初级抗体溶液中的视网膜。视网膜应在任何时候都充分浸渍在150至200微升的溶液。 </li><li>稀释的驴抗山羊IgG二级抗体为2微克/毫升含2％的Triton X-100的PBS孵育视网膜在该溶液中2小时在RT轻轻摇动。视网膜应充分浸渍于在所有时间该抗体溶液。用铝箔覆盖的其余步骤，以防止光漂白的培养板。 </li><li>用刷子，视网膜仔细转移到幻灯片，使得组织位于平如POSsible。空气干燥10分钟。安装使用防褪色的安装介质。 </li><li>检查在荧光显微镜下视网膜平板坐骑。如所述量化每视网膜象限三个非重叠区域Brn3a阳性RGC的数目。18 </li></ol> 6.研资局轴突的量化对视神经横断面<ol><li>收集来自步骤4.5视神经和在2％四氧化锇2小时孵育（OSO 4，MW254.23克/摩尔）。 注意:由于其高毒性，四氧化锇应与合适的实验室装在通风橱处理。 </li><li>通过在每个15分钟增加的乙醇浓度（50％，70％，90％，95％和100％），浸渍脱水视神经。 </li><li>使用下列配方制备的环氧树脂:15.72毫升嵌入-812，6.45毫升十二碳烯基琥珀酸酐，7.83毫升纳迪克甲基酸酐，0.45毫升DMP-30。 </li><li>依次在孵化解决方案组成的视神经0:1，1:1，0.75:0.25和1:环氧树脂的下列比率环氧丙烷（分子量58.08克/摩尔）的0。视神经在RT每个解决方案为O / N期培养。 </li><li>孵育嵌入在60℃下100％环氧树脂（从6.4最后步骤）另外48小时视神经。 </li><li>使用切片机生成半薄视神经截面（0.75微米）。 </li><li>染色用1％甲苯胺蓝视神经横截面。 </li><li>量化RGC轴突在每个视神经节描述19五个非重叠区域。 </li></ol>

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H., Hernandez, M., Vecino, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+experimental+glaucoma+models+in+rats:+comparison+of+the+effects+of+intraocular+pressure+elevation+on+retinal+ganglion+cell+size+and+death.">Three experimental glaucoma models in rats: comparison of the effects of intraocular pressure elevation on retinal ganglion cell size and death.</a> Exp Eye Res. 83, 429-437 (2006).</li><li>Weber, A. J., Zelenak, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+glaucoma+in+the+primate+induced+by+latex+microspheres.">Experimental glaucoma in the primate induced by latex microspheres.</a> J neuroscience meth. 111, 39-48 (2001).</li><li>Ho, L. C., 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=In+vivo+assessment+of+aqueous+humor+dynamics+upon+chronic+ocular+hypertension+and+hypotensive+drug+treatment+using+gadolinium-enhanced+MRI.">In vivo assessment of aqueous humor dynamics upon chronic ocular hypertension and hypotensive drug treatment using gadolinium-enhanced MRI.</a> Invest Ophthalmol Vis Sci. 55, 3747-3757 (2014).</li><li>Yang, Q., 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=Microbead-induced+ocular+hypertensive+mouse+model+for+screening+and+testing+of+aqueous+production+suppressants+for+glaucoma.">Microbead-induced ocular hypertensive mouse model for screening and testing of aqueous production suppressants for glaucoma.</a> Invest Ophthalmol Vis Sci. 53, 3733-3741 (2012).</li><li>Gallego, B. I., 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=IOP+induces+upregulation+of+GFAP+and+MHC-II+and+microglia+reactivity+in+mice+retina+contralateral+to+experimental+glaucoma.">IOP induces upregulation of GFAP and MHC-II and microglia reactivity in mice retina contralateral to experimental glaucoma.</a> J neuroinflammation. 9, 92 (2012).</li><li>Rojas, B., 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=Microglia+in+mouse+retina+contralateral+to+experimental+glaucoma+exhibit+multiple+signs+of+activation+in+all+retinal+layers.">Microglia in mouse retina contralateral to experimental glaucoma exhibit multiple signs of activation in all retinal layers.</a> J neuroinflammation. 11, 133 (2014).</li><li>Nadal-Nicolas, F. M., 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=Brn3a+as+a+marker+of+retinal+ganglion+cells:+qualitative+and+quantitative+time+course+studies+in+naive+and+optic+nerve-injured+retinas.">Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas.</a> Invest Ophthalmol Vis Sci. 50, 3860-3868 (2009).</li><li>Morquette, B., 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=REDD2-mediated+inhibition+of+mTOR+promotes+dendrite+retraction+induced+by+axonal+injury.">REDD2-mediated inhibition of mTOR promotes dendrite retraction induced by axonal injury.</a> Cell Death Differ. 22, 612-625 (2015).</li><li>Almasieh, M., Zhou, Y., Kelly, M. E., Casanova, C., Di Polo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+neuroprotection+in+glaucoma:+role+of+galantamine-mediated+activation+of+muscarinic+acetylcholine+receptors.">Structural and functional neuroprotection in glaucoma: role of galantamine-mediated activation of muscarinic acetylcholine receptors.</a> Cell Death Dis. 1, 27 (2010).</li></ol>

The authors declare that they have no competing financial interests.

这里介绍的视频技术提供关于如何执行磁珠的房内注射，有效和可重复诱发眼压升高小鼠详细的一步一步的指示。在持续眼压增加，不需要额外的注射和促进第3周高眼压induction.Elevated眼压内检测RGC胞体和轴突损失这个过程的结果是在人类发展青光眼的主要危险因素。因此，这是一个有价值的鼠眼压依赖性青光眼具有用于广泛的应用中潜在模式。 与微珠的注射有关进入前房的共同缺点涉及通过注射点对珠粒回流下当针被撤回，这经常导致在含水流出并增加变异仅部分阻塞。为了解决这个问题，一些重要的修改得到落实。杉杉<html> T，具有多面斜角干净，锋利的玻璃微针的精心准备是微珠的成功注入必不可少。一个正确编写微针能随压力最小的应用细腻的眼球表面的角膜受控流畅渗透。小角膜穿刺防止微球的回流。此外，细微针减少破坏性附近结构如虹膜和晶状体，这可能导致在非疾病相关的炎症的风险。其次，中，注射后手持磁铁战略眼部领域的应用是这项技术的另一个重要方面。在注射过程中，磁体用于当微针缩回到磁微珠绘制到微珠前房防止回流。注射后，磁体然后用于引导微珠的虹膜角膜角到方框房水外流。 帐篷"&gt;在微珠闭塞模型中经常遇到的另一个问题是，重复珠注射常常有必要实现持续IOP升高10,11。这可能是微珠从虹膜角膜角撞出随时间。手持磁体的组合的结果，如上所述，和鼠标的定位手术后大大提高的结果。使用注射麻醉剂，这允许灵活地在操作过程中移动至头部和需要较长的手术后的恢复期，是有利的。的放置鼠标与操作眼朝上为外科手术后的几个小时有助于在虹膜角膜角的结算微珠和减小撞出回进入前房的风险。 确保注入小珠的数量是相对一致的是最小化的动物间变异另一个关键步骤。由于微珠定居在Bottom管，有必要充分均质化微珠溶液和撤回适当体积进入微针及时。少珠粒喷射进入前房可能导致房水排水结构的不完全堵塞，这可能导致较差的或可变的IOP升高。值得注意的是，虽然微珠注入的最终目的是提高眼压，应谨慎时从清醒小鼠眼压测量比在这项研究中（〜25毫米汞柱）报告峰值更高服用。极高的IOP增加缺血损伤的风险，并且还可能引起疼痛的动物。眼压的高度应被视为许多因素，以评估手术的成功之一。因此，该过程的结果应当基于若干参数，包括高眼压，RGC体躯死亡，轴突损失来衡量。 虽然这里所描述的协议，导致成功的最微珠LY沉降以角度，该模型的一个潜在的局限性是，那些仍然在前房漂珠可能与通过角膜活视网膜成像，以及需要的光的有效通道电或行为测定干扰。另一个重要的方面来考虑，当利用该微珠闭塞模型是高眼压和随后RGC变性的程度与所​​操作的鼠标[4]的年龄和遗传背景而变化。因此，就需要为每个特定的转基因小鼠品系和/或年龄范围被确定IOP升高的程度和RGC变性的时间轴。 这种模式的一个特点是微珠注射，并显著RGC死亡后的前三个星期在RGC死亡的逐渐丧失高眼压的结果是在手术后3周检测。因此，这种模式使的发生在该D早期和/或微妙的变化检查isease，之前公开的RGC胞体和轴突的损失。在RGC死亡一显著增加并不高眼压诱导后3和6周的观察。尽管成功3和6周之间25％的和持续的高眼压在这些时间点 - 事实上，RGC胞体和轴突损失在〜22保持稳定。可能需要持续的眼压持续时间较长的额外RGC损失C57BL发生/ 6小鼠，这似乎相比其他小鼠品系要到RGC的损害更耐。5其他修改，这里介绍的协议，其中包括珠大小的调整和额外的注射剂，可能需要以研究在稍后的时间点的RGC损失。因此，我们的协议是理想的集中在适度的RGC神经退行性这是有关发生和早期发展在人类青光眼早期相关的病理生理变化的研究。

青光眼是一种渐进的和不可逆的致盲条件下，将在2020年1影响全世界估计有8000万人。在青光眼患者，视力丧失是由视网膜神经节细胞（RGC），从传递视觉信息的输出神经元选择性死亡引起的视网膜到大脑。青光眼是年龄相关的神经变性疾病的许多危险因素，其中最常见的是升高的眼内压（IOP）。事实上，眼压是唯一可改变的危险因素和青光眼的治疗目前只专注于管理眼压。然而，多个基因，细胞，和环境因素影响这种疾病的发作和进展。因此，了解，最终有助于神经元死亡的各种机制是开发用于治疗青光眼的有效治疗是至关重要的。 青光眼的动物模型是研究疾病的病理生理学，并确定和试验的基本要求有前途的治疗。转基因小鼠线，包括条件性敲除品系小鼠携带的基因编码荧光示踪剂的日益普及，推动了可诱导小鼠模型青光眼的需要。青光眼的几个啮齿动物模型已经发展了很多年（以2,3综述）。在许多这样的车型，青光眼是通过破坏房水动力学，导致眼压的升高所致。闭塞模型，其中微珠或其他物质被注入到眼睛的前房阻止水排水，近年来部分由于其相对容易增加眼压4-14得到普及。 青光眼的微珠闭塞模型，首次在灵长类动物中12进行，兔8，和老鼠4,9,11，最近被改编为小鼠5,6,10使用。在这些研究中，聚苯乙烯微珠的房内注射，单独使用或以用粘弹性材料组合，导致IOP升高导致随后的RGC死亡6,10。然而，当针从虹膜角膜角微珠的眼睛和撞出抽出回流是在操作过程中出现的常见问题。为了尽量减少这些缺点，磁铁已用于吸引磁微珠到眼睛4,9的虹膜角膜角。 此处所描述的协议是基于以前的研究9,10使用磁微珠和手持磁体适于鼠标眼（ 图1）的改性方法。一些重要的修改已经在我们的协议被引入，以确保在小鼠有效和可重复的眼压升高。首先，微珠的注入用精心准备的玻璃微针与刻面斜角完成。微针的所得光滑的表面以及其锋利的尖端可确保最小的损害是造成它刺穿角膜。使用这种玻璃微针还导致增加的控制时，微型针尖端进入前房，从而减少有害附近结构的风险，如虹膜和晶状体。另外，微小的注射病变促进角膜自我修复，并减少不必要的损伤相关的影响。 第二，磁微珠的注射和使用手持磁体允许精确控制，以吸引小珠的虹膜角膜角的小鼠眼。磁微珠是4.5微米的直径被使用，因为这种微珠大小没有堵塞制备微针开口和重要的是，一旦注射，这些微珠有效阻止房水的排出。这种方法不仅降低了注射微珠回流下，也确保了微珠的最大数量的目标区域累积到有效阻挡房水引流。 Furthermore，这种策略也减少了珠漂浮在前房避免与其他结构，如虹膜​​和晶状体，并后房防止通道接触的数目。总的来说，这些修改确保微珠注入手术相对容易和在导致高眼压在小鼠中高度重现，有效和持久的感应及时执行。

<table>
	<tbody>
		<tr>
			<td>Puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin Wall Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>TW150F-4</td>
			<td>Capillary has an outer diameter of 1.5 mm and inner diameter of 1.12 mm</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Zeiss</td>
			<td>MZ9.5</td>
			<td>Zoom factor range of 2.5 to 6.0. Microscope used for needle-making and the micro-bead injection surgery.</td>
		</tr>
		<tr>
			<td>Footswitch</td>
			<td>Linemaster</td>
			<td>T-91-SE</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Blade</td>
			<td>Feather</td>
			<td>No. 11</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Beveler</td>
			<td>Science Products</td>
			<td>BV-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Duster</td>
			<td>Fisher</td>
			<td>23-022-523</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>BP359-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientific</td>
			<td>12-812</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads M-450 Epoxy</td>
			<td>Life Technologies</td>
			<td>14011</td>
			<td>Magnetic beads are 4.5 &#181;m in diameter. Stock solution is at a concentration of 4 x 108 beads/mL. Store at 4&#176;C.</td>
		</tr>
		<tr>
			<td>Mini-Tube Rotators</td>
			<td>Fisher Scientific</td>
			<td>05-450-127</td>
			<td></td>
		</tr>
		<tr>
			<td>3 Handheld Magnets</td>
			<td>Geomag</td>
			<td></td>
			<td>0.45 Tesla. Magnet used for microbead preparation and microbead injection surgery.</td>
		</tr>
		<tr>
			<td>25 mL serological pipet</td>
			<td>Costar</td>
			<td>4489</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet</td>
			<td>Drummond</td>
			<td>4-000-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological Containment Hood</td>
			<td>Biostad</td>
			<td>377355</td>
			<td></td>
		</tr>
		<tr>
			<td>Balanced salt solution (BSS)</td>
			<td>Alcon</td>
			<td>0065-0800-25</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 Micropipet</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtube 1.5 mL</td>
			<td>Sarstedt</td>
			<td>72.690</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 Micropipet</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 mL PCR tube</td>
			<td>Sarstedt</td>
			<td>72737.002</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td></td>
			<td></td>
			<td>Controlled substance</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Vetoquinol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U-100 Insulin Syringe</td>
			<td>Becton Dickinson and Company</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Ohaus</td>
			<td>CS 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td></td>
			<td></td>
			<td>Controlled substance</td>
		</tr>
		<tr>
			<td>Tropicamide ophthalmic solution</td>
			<td>Alcon</td>
			<td>0998-0355-15</td>
			<td>1% Mydriacyl</td>
		</tr>
		<tr>
			<td>Manual Microsyringe Pump with Digital Display</td>
			<td>World Precision Instruments</td>
			<td>DMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>M3301R</td>
			<td></td>
		</tr>
		<tr>
			<td>Platform</td>
			<td>Fisher Scientific</td>
			<td>14-673-52</td>
			<td>8 x 8 inch</td>
		</tr>
		<tr>
			<td>Absorbent swabs</td>
			<td>Kettenbach</td>
			<td>30601</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 Micropipet</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic forcep</td>
			<td>Euroband</td>
			<td>1001</td>
			<td>Ensure forcep is plastic and has a flat surface to avoid damaging the eye</td>
		</tr>
		<tr>
			<td>Fluoroquinolone ophthalmic solution</td>
			<td>Alcon</td>
			<td></td>
			<td>Vigamox</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Sunbeam</td>
			<td>E12107-834</td>
			<td></td>
		</tr>
		<tr>
			<td>Tonometer</td>
			<td>iCare</td>
			<td>TV02</td>
			<td>TONOLAB rebound tonometer&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, Para</td>
			<td>Fisher Scientific</td>
			<td>T353-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde solution</td>
			<td>Sigma-Aldrich</td>
			<td>G7651</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacodylate, tryhydrate</td>
			<td>Canemco and Marivec</td>
			<td>124-65-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Brn-3a antibody (C-20)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-31984</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Plate, 48 well</td>
			<td>Falcon</td>
			<td>353078</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate</td>
			<td>Life Technologies</td>
			<td>A-11058</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Slow fade Gold antifade reagent</td>
			<td>Life Technologies</td>
			<td>S36936</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass</td>
			<td>Fisher Scientific</td>
			<td>12-548-5E</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide 2% aqueous solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>3294949</td>
			<td></td>
		</tr>
		<tr>
			<td>Embed-812</td>
			<td>Electron Microscopy Sciences</td>
			<td>14900</td>
			<td></td>
		</tr>
		<tr>
			<td>Dodecenyl succinic anhydride</td>
			<td>Electron Microscopy Sciences</td>
			<td>13710</td>
			<td></td>
		</tr>
		<tr>
			<td>Nadic methyl anhydride</td>
			<td>Electron Microscopy Sciences</td>
			<td>19000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP-30</td>
			<td>Electron Microscopy Sciences</td>
			<td>13600</td>
			<td></td>
		</tr>
		<tr>
			<td>Propylene oxide</td>
			<td>Sigma-Aldrich</td>
			<td>110205-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding mold-Dykstra</td>
			<td>Electron Microscopy Sciences</td>
			<td>70907</td>
			<td></td>
		</tr>
		<tr>
			<td>Porter-Blum ultra-microtome</td>
			<td>Sorvall</td>
			<td>MT-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue O (Certified Biological Stain)</td>
			<td>Fisher-Scientific</td>
			<td>T161-25</td>
			<td></td>
		</tr>
	</tbody>
</table>

a magnetic microbead occlusion model to induce ocular hypertension-dependent glaucoma in mice

使用青光眼的啮齿动物模型一直必须了解背后这种多因素的神经变性疾病的病理生理学的分子机制。与许多转基因小鼠系的出现，有在高眼压诱导小鼠模型越来越大的兴趣。这里，我们提出基于使用改性微针具有刻面斜角喷射磁微珠的进入眼睛的前房青光眼的闭塞模型。磁微珠被吸引到使用手持磁体阻止房水从前房排水的虹膜角膜角。这种破坏在眼内压，这随后导致视网膜神经节细胞的损失，如在人类青光眼患者中观察到的稳定的标高含水动力学的结果。在这个手稿提出的微珠闭塞模型比较简单青光眼等可诱导模型，也高度有效和可重复性。重要的是，这里提出的修改最小化，往往发生在闭塞模型的常见问题。首先，使用一个斜面玻璃微针的防止微珠的回流，并确保在注射过程中发生的角膜损伤最小，从而减少损伤相关的影响。第二，利用磁微珠的确保吸引大多数珠粒的虹膜角膜角，从而有效地减少了珠漂浮在前房避免与其他结构的接触（ 例如 ，虹膜，透镜）的数目的能力。最后，使用手持磁体允许灵活性处理小鼠眼有效地引导磁微珠并确保有从眼睛微珠的小回流时该微针被撤回时。总之，这里提出的微珠闭塞小鼠模型是一个功能强大的调查工具，研究glau的发作和进展过程中发生的神经变性昏迷。

磁微珠的注入在该协议描述成年小鼠的前房导致眼压的一个强大的和可再现的高程。一个星期后的程序，眼压从10±0.6毫米汞柱（平均值±SEM），对侧眼的平均眼压基线高血压眼睛增加，19±0.5毫米汞柱（学生t检验; *** P &lt;0.001， N = 12， 表1，图2）。眼压此后稳定，并在为至少6周，在本研究检查了最长的时间点上的平均为20毫米汞柱的仍然升高。平均峰值眼压微珠注入眼睛2,3和6周手术后为25毫米汞柱。绝大多数治疗的小鼠的发展持续高眼压，因此该协议不需要微珠第二次注射。 为了评估该模型RGC损失的时间过程，RGC躯体均首先通过与Brn3a，特定RGC标记物17免疫染色定量。 Brn3a阳性细胞的数目上的平面安装的视网膜的眼内高压诱导后在1，2，3和6周定量。虽然微珠注射后早在第1周时检测到显著IOP升高，是在第2周的程序（ 图3）的内观察到RGC胞体的无显著损失。实质性RGC死亡（22％），然而，在第3周明显（2,430±67的RGC / mm 2时，平均±SEM，N = 12）和6周（2350±74的RGC 个 / mm 2中，n = 10）后感应的高眼压症，与从非手术小鼠（3,141±49的RGC 个 / mm 2中，n = 23）（ANOVA，p &lt;0.001）完整对照眼。 研资局轴突功能障碍和变性是青光眼的一个重要特征。因此，轴突损失在3和6周微珠注射后观察在用甲苯胺蓝（ 图4）染色视神经截面RGC轴突的定量。 3周观察RGC轴突的大幅亏损（25％）（28401±702轴突/神经，平均值±SEM，N = 5）和6周（29426±948轴突/神经，N = 6）微球注射后与从非手术的眼睛完好视神经（39,467±137轴突/神经，N = 4）（ANOVA，p &lt;0.001）。总的来说，这些数据表明，注射磁微珠的入小鼠前房导致可重复的和持续的高眼压，其导致RGC胞体和轴突变性。 <table><tbody><tr><td colspan="2"> OHT手术后时间 </td><td> ñ </td><td colspan="3"> 平均眼压（毫米汞柱）±SEM </td><td colspan="2"> 峰值眼压（毫米汞柱） </td></tr><tr><td></td><td></td><td></td><td>对侧</td><td>青光眼</td><td>迪fference </td><td>对侧</td><td>青光眼</td></tr><tr><td colspan="2"> 1周</td><td> 12 </td><td> 10±0.4 </td><td> 19±0.5 </td><td> 9±0.6 </td><td> 12±0.4 </td><td> 22±0.6 </td></tr><tr><td colspan="2"> 2周</td><td> 13 </td><td> 11±0.5 </td><td> 20±0.8 </td><td> 9±0.5 </td><td> 12±0.9 </td><td> 25±0.7 </td></tr><tr><td colspan="2"> 3周</td><td> 10 </td><td> 11±0.8 </td><td> 20±0.7 </td><td> 10±0.9 </td><td> 13±0.2 </td><td> 25±0.9 </td></tr><tr><td colspan="2"> 6周</td><td> 12 </td><td> 12±0.5 </td><td> 20±0.6 </td><td> 9±0.7 </td><td> 13±0.5 </td><td> 24±0.6 </td></tr></tbody></table> 表1.鼠磁微珠Occlusi眼压升高在模型。在清醒的女性C57 BL / 6小鼠，眼压使用校准反弹眼压计测量。操作眼睛显示1周手术后检测眼压增加，这一程序后至少六周仍然升高。 <img alt="图1" src="/files/ftp_upload/53731/53731fig1.jpg" /> 图1.工作流青光眼的鼠磁微珠缺血模型涉及的步骤的。期间之前进行的所有过程一步一步的轮廓，并且在手术后。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53731/53731fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53731/53731fig2.jpg" /> 图2.眼内压升高在小鼠磁微珠缺血模型，在清醒雌性C57 BL / 6小鼠，眼压使用校准反弹眼压计测定的。微珠注入眼睛的眼压1周后的外科手术（ANOVA，P &lt;0.001）的显著升高。 IOPS的仍然显著为相对至少6周（ANOVA，P &lt;0.001）提升到注入小鼠的对侧眼。 （完整:N = 12;第1周:N = 12，2周:N = 13，2周:N = 10 6周:N = 12）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53731/53731fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53731/53731fig3.jpg" /> 图3.在小鼠磁微珠闭塞模型。RGC的视网膜神经节细胞死亡通过在完整控制视网膜（A）和3周和6周青光眼视网膜使用Brn3a平面安装的视网膜免疫染色微珠注射后可视化以诱导高眼压症（OHT）（B，C）。比例尺:20μm左右。 （D）的定量分析证实微珠注入在3和6周导致显著RGC索玛损失相比，控制的眼睛后的程序。在完整的，非青光眼C57 / BL6小鼠的RGC体细胞的密度被示为参考（白色条，100％存活）。值表示为平均值±SEM（完整:N = 23;第1周:N = 6，2周:N = 6，3周:每组12 6周:每组10只，ANOVA，*** P &lt; 0.001）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53731/53731fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53731/53731fig4.jpg" /> 图4.轴突变性中的鼠磁微珠闭塞模型。RGC轴突由视神经截面染色用甲苯胺蓝在可视化微珠注射后的完整控制（A）和青光眼视网膜在3和6周，诱导高眼压症（OHT）（B，C）。比例尺:10μm以下。 （D）的定量分析证实微珠注入在3和6周导致显著RGC轴突损失相比，控制的眼睛后的程序。在完整的，非青光眼C57 / BL6小鼠的RGC轴突的密度被示为参考（白色条，100％存活）。值表示为平均值±SEM（完整:N = 4;3周:N = 5 6周:N = 6，方差分析，*** P &lt;0.001）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53731/53731fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice at JoVE.com

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

A磁条微珠缺血模型，以诱导小鼠高眼压，青光眼相关

在这里，我们提出了一个协议，以诱发高眼压在小鼠眼睛中青光眼观察到，导致视网膜神经节细胞的损失。磁微珠被注入到前房并使用磁体来阻止房水的流出吸引到虹膜角膜角。

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

a-magnetic-microbead-occlusion-model-to-induce-ocular-hypertension

Nanyang Technological University

Research

JoVE Journal

Medicine

19.2K 次观看.  CRCHUM and University of Montreal. 在这里，我们提出了一个协议，以诱发高眼压在小鼠眼睛中青光眼观察到，导致视网膜神经节细胞的损失。磁微珠被注入到前房并使用磁体来阻止房水的流出吸引到虹膜角膜角。 

视频: A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

建模在小鼠体内的行程 - 中东与灯丝模型大脑中动脉闭塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

科研

医学

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

压力控制通气诱导小鼠急性肺损伤

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important 7, 14, 30, 39. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.
Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP 39. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.
We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds39. In addition to infarct staining, we tested cardiac troponin I (cTnI)
as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction6, 8, 30, 40, 41. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.

A murine model for myocardial ischemia and ischemic preconditioning is an important tool study cardioprotective mechanisms in vivo. Here, we report an easy applicable in situ model for cardiac IP using a hanging-weight system for coronary artery occlusion.

悬重系统的使用在小鼠冠状动脉闭塞

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available 1-38. ...

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

CYP2D6动物模型:如何诱导小鼠自身免疫性肝炎

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

双侧颈总动脉闭塞足够的预处理刺激诱导局灶性脑缺血早期缺血耐受

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

可重放截瘫胸主动脉阻断脊髓缺血再灌注的小鼠模型

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

实验诱发青光眼通过磁性微球注射眼

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

青光眼诱导在程序<em&gt;在体内</em&gt;大鼠模型和全安装视网膜准备

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

对大脑中动脉闭塞再灌注损伤诱发中风小鼠的手术方法

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

诱导小鼠横向主动脉狭窄的闭合胸模型

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...