We wish to thank Peter Munro PhD for his assistance with optic nerve sectioning. This study was supported by the Medical Research Council (G0901303), and in part by the Dorothy Hodgkin Postgraduate Award/Medical Research Council, the Helen Hamlyn Trust, Fight for Sight, and Moorfields special trustess,.

伦理学声明 :所有的动物实验均按照该机构动物护理和使用委员会（IACUC）已经进行，并批准了与英国内政部的指导方针一致（ <a href="http://goo.gl/FLkirW" target="_blank">http://goo.gl/FLkirW</a> ，上次访问10 日止 ，2014年）和ARVO声明动物的眼科和视觉研究（使用<a href="http://goo.gl/4LFOjD" target="_blank">http://goo.gl/4LFOjD</a> ，上次访问10 日 2014年6月，）。 1.高眼压感应<ol><li>通过提高经由单侧注射顺磁性微球的眼内压（IOP）到棕色挪威大鼠前房，基于Samsel 等人的方法中诱导实验性青光眼。3的其它着色大鼠可以是合适的，尽管这些将需要进行验证首先由用户设定。 </li><li>楼250-300克重的雌前饲养员布朗Norway大鼠中的恒定的低光环境（40-60勒克斯），以在眼压7最小化昼夜的波动，以获得食物和水随意 。 </li><li>就拿基线眼压测量清醒动物8前麻醉和注射珠，利用反弹眼压计校准大鼠的眼睛9使用。眼压取为5个读数的平均值。 </li><li>麻醉大鼠37.5毫克/公斤氯胺酮，并发表腹腔0.25毫克/公斤盐酸美托咪定。通过测试动物的后足反射确定麻醉深度，聚维酮碘的应用程序（见步骤1.5），和珠注射之前（见步骤1.8）。管理0.5％盐酸丙美卡因镇痛。 注:在任何阶段不要扩张瞳孔。这将有助于珠粒沉降更好入虹膜角膜角，并防止结合到透镜。适用于眼软膏以防止非手术对侧眼的角膜干燥。 </li><li> W¯¯灰手术眼用5％聚维酮碘在水注射前10 5分钟。 </li><li> 5分钟后，灯芯的聚维酮碘断用无菌纱布，并用0.9％无菌盐水的溶液洗眼睛。保持眼部麻醉用无菌生理盐水常规应用过程中湿润。 </li><li>将眼睛周围的环形磁铁。 </li><li>注入25μl的含30毫克/毫升的γ-照射消毒的Hank氏平衡盐溶液（HBSS）8微米的磁性微球进入前房，用33克斜口针的溶液中。 <ol><li>为了制备珠，通过再悬浮洗涤，然后离心3次以10,000×g离心5分钟，用1ml HBSS，使最后的30毫克/毫升溶液之前。保持整个无菌条件。 </li><li>对于注射，要小心避免插入针虹膜，尽量减少虹膜损伤的风险。这可以通过定向针正切于角膜表面被防止，为平行于虹膜越好。这也将有助于减少从注射部位珠损失。 注:注入珠以极快的速度，以确保均匀围绕虹膜角膜角，这是为了提高眼压关键分布。此外，存储珠，针头和磁性环分开，使得所述珠不形成簇，使得它们难以加载到注射器中，并注入，以及针不会被磁化。 </li></ol></li><li>留针到位1分钟后注射，以确保珠融入了房角从小梁阻碍房水引流。稍后角珠针已初步解决，使水的一些泄漏，最大限度地减少瞬时增加IOP。在手术结束时通过冲洗将针与第一磷酸盐缓冲盐水（PBS），然后70％的乙醇，随后用蒸馏水，以确保继续使用在分开的程序针。可替代地，人们可以使用如果正确的计和注射器组合是可用的一次性针头。可选:针可以使用beveller延长它们的使用被激化。 </li><li>在此阶段，如果需要，除去磁体，并用它来绘制珠粒成不完全覆盖的区域。 </li><li>离开磁铁在眼睛周围的地方为另外的10分钟后喷射，确保珠粒沉降顺利进入虹膜角膜角。 </li><li>使用0.25毫克/公斤盐酸反atipemezole麻醉。 </li><li>辖氯霉素，或其他抗生素软膏，例如庆大霉素或土霉素局部，以防止感染，和0.5％的盐酸丙美卡因镇痛。离开动物恢复上热垫，直到他们恢复运动，然后转移到一个暖箱和供应额外的营养，比如辅食或蘸正常饮食，直到恢复完成。全身或局部镇痛应给动物手术后疼痛显示24小时的迹象。如果THESË症状持续，尽管治疗，动物应得到人道扑杀。 </li><li>用对侧眼作为非工作的控制。 </li><li>就拿IOP测量每2-3天珠给药后，每2-3天使用后的反弹眼压计校准大鼠的眼睛9使用。 </li><li>的标准，包括眼睛的研究中可以是:如果1）眼压升高对侧控制压力之上5毫米汞柱，和2）不超过60毫米汞柱。 </li><li>眼睛其中压力恢复到基线（通常1周后喷射）不应该被包括在研究中，但也可以在眼睛失败，如果需要开发高眼压重新注入珠。 </li><li>安乐死的动物用CO 2窒息在实验结束。 </li><li>解剖眼睛和视觉神经，并在4％多聚甲醛（PFA）过夜进一步的组织学分析解决。 </li></ol> 2.评估视网膜神经元损伤使用TUNEL S泰宁<ol><li>为了量化凋亡细胞在整装retinasuse终端脱氧核苷酸转移酶介导的dUTP缺口标记（TUNEL）测定中，根据制造商的说明。 </li><li>解剖从眼罩视网膜，冲洗3×5分钟，在0.3％的Triton X-100的磷酸盐缓冲盐水（T-PBS）中。 </li><li>透在3％的T-PBS中的组织2小时。 </li><li>在37℃预平衡的平衡缓冲液视网膜10分钟，温育在TUNEL反应溶液之前1小时。 </li><li>洗净组织在0.3％T-PBS 3×5分钟，冲洗0.3％T-PBS含5μMDAPI，并在安装媒体平板安装。 </li><li>量化TUNEL阳性细胞核使用共聚焦显微镜拍到10微米的z栈通过神经节细胞层，在20X放大倍数。取3个图像上每个4花瓣，在站点靠近视神经，在中间周，并在视网膜的远边缘，总计每整MOUN 12的图像T，采样约7000细胞。形态学标准区分非神经元（内皮细胞和神经胶质细胞），从神经元细胞。 </li><li>仅使用DAPI通道，并且掩模研究者对治疗组进行摄像选择区域。 </li></ol> 3.评估视神经损害使用​​甲苯胺蓝染色<ol><li>修复视神经一夜之间在4℃Karnovsky的解决方案。 </li><li>在1％（重量/体积）的四氧化锇处理试样2小时，然后脱水在100％乙醇。 </li><li>孵育视神经中的氧化丙烯30分钟，并将其放置在环氧丙烷的50:50混合物:ARALDITE过夜。 </li><li>过夜，在60℃下改变该溶液至100％ARALDITE，接着培养。 </li><li>切半薄切片（0.75毫米厚），并染色，用1％甲苯胺蓝/硼​​砂（TB）的在50％乙醇考试之前通过光学显微镜。 </li></ol> 4.统计分析<ol><li>进行统计语录裂解用适当的统计软件包。双向ANOVA和纽曼-Keul的事后检验可以用于计算统计学显着性为眼压随时间而改变。 </li><li>小于0.05的p值可以被认为是显著。 </li></ol>

<ol>
	<li>Quigley, H. A., Broman, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+number+of+people+with+glaucoma+worldwide+in+2010+and+2020.">The number of people with glaucoma worldwide in 2010 and 2020.</a> Br J Ophthalmol. 90 (3), 262-267 (2006).</li><li>Weinreb, R. N., Aung, T., Medeiros, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+and+treatment+of+glaucoma:+a+review.">The pathophysiology and treatment of glaucoma: a review.</a> JAMA. 311 (18), 1901-1911 (2014).</li><li>Samsel, P. A., Kisiswa, L., Erichsen, J. T., Cross, S. D., Morgan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+induction+of+experimental+glaucoma+using+magnetic+microspheres.">A novel method for the induction of experimental glaucoma using magnetic microspheres.</a> Invest Ophthalmol Vis Sci. 52 (3), 1671-1675 (2011).</li><li>Libby, R. 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=Inherited+glaucoma+in+DBA/2J+mice:+pertinent+disease+features+for+studying+the+neurodegeneration.">Inherited glaucoma in DBA/2J mice: pertinent disease features for studying the neurodegeneration.</a> Vis Neurosci. 22 (5), 637-648 (1017).</li><li>Morrison, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+intraocular+pressure+and+optic+nerve+injury+models+in+the+rat.">Elevated intraocular pressure and optic nerve injury models in the rat.</a> J Glaucoma. 14 (4), 315-317 (2005).</li><li>Levkovitch-Verbin, H., 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=Translimbal+laser+photocoagulation+to+the+trabecular+meshwork+as+a+model+of+glaucoma+in+rats.">Translimbal laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats.</a> Invest Ophthalmol Vis Sci. 43 (2), 402-410 (2002).</li><li>Moore, C. G., Johnson, E. C., Morrison, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+rhythm+of+intraocular+pressure+in+the+rat.">Circadian rhythm of intraocular pressure in the rat.</a> Curr Eye Res. 15 (2), 185-191 (1996).</li><li>Morrison, J. C., Jia, L., Cepurna, W., Guo, Y., Johnson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+and+sensitivity+of+the+TonoLab+rebound+tonometer+in+awake+Brown+Norway+rats.">Reliability and sensitivity of the TonoLab rebound tonometer in awake Brown Norway rats.</a> Invest Ophthalmol Vis Sci. 50 (6), 2802-2808 (2009).</li><li>Wang, W. H., Millar, J. C., Pang, I. H., Wax, M. B., Clark, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+measurement+of+rodent+intraocular+pressure+with+a+rebound+tonometer.">Noninvasive measurement of rodent intraocular pressure with a rebound tonometer.</a> Invest Ophthalmol Vis Sci. 46 (12), 4617-4621 (2005).</li><li>Isenberg, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ocular+application+of+povidone-iodine.">The ocular application of povidone-iodine.</a> Community Eye Health. 16 (46), 30-31 (2003).</li><li>Morrison, J. 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=A+rat+model+of+chronic+pressure-induced+optic+nerve+damage.">A rat model of chronic pressure-induced optic nerve damage.</a> Exp Eye Res. 64 (1), 85-96 (1997).</li><li>Foxton, R. H., 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=VEGF-A+is+necessary+and+sufficient+for+retinal+neuroprotection+in+models+of+experimental+glaucoma.">VEGF-A is necessary and sufficient for retinal neuroprotection in models of experimental glaucoma.</a> Am J Pathol. 182 (4), 1379-1390 (2013).</li></ol>

David T. Shima is an employee of Hofmann-La Roche, Switzerland. The remaining authors declare they have no competing financial interests.

在这里，我们证明用于诱导高眼压在大鼠，注射的磁性微球到眼睛的前房的方法。这种方法是简单的开展，并要求小的手术经验，或实践和细化小时。此外，该过程是有效的;很少需要多于单次注射的珠诱导强，健壮的压力升高（约10％的回注率）。这可以提供优于现有诱导方法，如在技术上具有挑战性episceral静脉硬化11模型，或激光光凝协议6，它可以要求以维持升高的IOP的多个程序。 为了不过是成功的方法中，有一些需要采取一些小的关键步骤。首先，它是非常有用的，以使用一个环形的磁铁吸引小珠到虹膜角膜角。这一步是原始协议，wher的变形E中的珠粒分别注入前房，然后移至徒手眼部周围3。使用环形磁铁意味着微球应该解决周围均匀的角度，需要最少的手动重新分配。其次，注入的速度要快 - 太慢和珠粒将主要积聚在角的一侧，导致不完整的覆盖，并有可能没有压力上升。一般来说，虽然，该方法是足够简单的，用户可以很容易地进行修改的协议，如改变微球粒的尺寸或体积，也许试图改变IOP升高的程度。 然而，该方法的一个潜在的缺点是一个具有很少的控制高血压，这在大约5-10％，我们观察到的情况下上升到超过60毫米汞柱的范围内。眼压过度升高可以很破坏视网膜组织，而且可能使学习机制和生物细胞死亡的迟缓挑战。然而，该方法产生一个一致的神经病理学，无论是在视网膜和视神经，其可药理学12被操纵。这可能使开发创新疗法用于治疗青光眼模型的吸引力。另外，由于珠引导到虹膜角膜角，这留下自由为视网膜或视神经乳头的实时成像的视轴。我们预计，这种模式将进行调整和用于其他物种未来的应用，包括鼠标。

原发性青光眼是一种破坏性的眼科疾病在世界各地1影响的估计6050万的人，这可能会导致生命改变的视力下降和失明2。研究这种疾病的机制，以及新的治疗青光眼的发展，是依赖于疾病的良好模型其中概括一些病理的标志。 我们在这里提出一个大鼠青光眼模型基于Samsel 等人的方法。3本技术的总的目标是通过注入磁性微球进入前房，并使用磁性环，直接增加的眼内压（IOP）的眼睛他们到虹膜角膜角。这阻碍了含水流出，从而增加了眼压，导致神经元损伤和细胞损失。该协议被开发，以试图提供青光眼的一个更简单的，可诱导的模型。 这个协议可以具有一些优点在现有的技术。可用，不需要的程序，启动基因小鼠模型，如DBA / 2J;然而，这些可能有不可预知的发病进展4。与此相反，可诱导的模型，其中大部分依赖于手术升降眼压在啮齿类动物，有引发可以由用户来控制的优点。其中一些方法可以具有它们自己的缺点然而，包括在技术上具有挑战性的5，并且可需要多个程序，以维持高眼压6。 与此相反，在该手稿详述的诱导方法是一种产生稳定的，坚固的增加的压力，以对回注最小需要简单，有效，和可重现的技术。此外，它不涉及昂贵的设备，并且仅需要基本的手术技巧来执行。该协议可能是适当的读者谁是寻求建立一个不太技术要求诱导青光眼模型在他们的实验室。

<table><tbody><tr><td>250 - 300g female Brown Norway ex-breeder rats</td><td>Harlan UK</td><td>203</td><td></td></tr><tr><td>Tonolab Rebound Tonometer</td><td>Tiolat</td><td>TV02</td><td></td></tr><tr><td>Ketaset (ketamine)</td><td>Fort Dodge Animal health</td><td>BN1000118</td><td>37.5 mg/kg</td></tr><tr><td>Domitor (medetomidine hydrochloride)</td><td>Orion Pharma</td><td>140-999</td><td>0.25 mg/kg</td></tr><tr><td>Povidone iodine</td><td>Ecolab</td><td>BN4369LE10</td><td>5% in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Minim&#039;s Saline Solution</td><td>Bausch and Lomb</td><td>PL00033/5017</td><td></td></tr><tr><td>Toroidal magnet</td><td>Supermagnete</td><td>R-10-07-03-N</td><td></td></tr><tr><td>Magnetic Microspheres</td><td>Bangs Laboratories</td><td>UMC4N/9692</td><td></td></tr><tr><td>HBSS</td><td>Invitrogen</td><td>14025</td><td></td></tr><tr><td>33 G beveled needle</td><td>Hamilton</td><td>7747-01</td><td>Custom needle</td></tr><tr><td>Luer tip syringe</td><td>Hamilton</td><td>80601</td><td></td></tr><tr><td>Antisedan (atipemezole hydrochloride )</td><td>Orion Pharma</td><td>141-003</td><td>0.25 mg/kg</td></tr><tr><td>Chloramphenicol ointment</td><td>Medicom</td><td>18956-0005</td><td></td></tr><tr><td>TUNEL staining kit</td><td>Promega</td><td>G3250</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Vectashield mounting media</td><td>Vector Labs</td><td>H-1000</td><td></td></tr></tbody></table>

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.

注射磁珠进虹膜角膜角一致诱导延长的和强大的压力升高（ 图1），这是很容易观察到，在第一时间点，3天注射后。此外，增加的压力保持在整个实验期间，虽然我们的时间过程完成18天注射后，其他人报告说，压力仍然存在长期3。平均眼压的平均值在实验进行控制的全长，非珠注射的眼中是19.7±0.3毫米汞柱，具有40.5±2.8毫米汞柱的胎圈注入眼（P &lt;0.001）。此外，峰值眼压从22.8±0.3毫米汞柱上升至49.9±2.3毫米汞柱。 以确定在眼压仰角是否导致视网膜神经节细胞的死亡，我们进行了TUNEL染色在视网膜，和组织学上横向视神经部分（ 图2）。在视网膜<html>我们观察到在珠注射的眼中的增加TUNEL染色（ 图2A）与高眼压。凋亡细胞的数量增加约15倍，从1.6±0.5细胞对侧控制，为24.5±0.5个细胞在高血压视网膜（ 图2B; P &lt;0.05）。此外，在眼，其中磁珠注射但压力没有增加（可能是由于虹膜角膜角的不完全堵塞），TUNEL阳性细胞的数量并没有显著不同于未注射的对照组（P&gt; 0.05）。这表明，细胞死亡有关的压力增加，而不是由于在磁性微球体直接毒性。最后，我们研究了视神经病变在青光眼模型，看见甲苯胺蓝的蓄积在许多轴突，表明这些细胞过程（ 图2C）的变性。 URE 1"SRC ="/文件/ ftp_upload / 52400 / 52400fig1highres.jpg"/&gt; 图1.高程的使用磁性微球体眼压升高。注射的磁性微球进入前房诱导健壮，显著升高的眼内压（IOP）相比，对侧对照，未注射的眼睛。 Y轴单位=毫米汞柱（毫米汞柱）。数据=平均值±SEM。 * = P &lt;0.001，N = 12，这个数字已经被修改福克斯顿等，Am。病理学杂志182（4）:1379年至1390年。 <img alt="图2" src="/files/ftp_upload/52400/52400fig2highres.jpg" /> 图2.高程眼压通过注射的磁性微球到虹膜角膜角，诱导的神经元死亡的神经节细胞层（GCL）。 （A）从控制（左）和青光眼（右）眼用TUNEL（绿色染色凋亡细胞核视网膜的代表图像;白色箭头）和DAPI（蓝色），表明凋亡细胞核的数量增加为眼压上升。（B）中的TUNEL阳性细胞的GCL定量，显示出眼高眼压（中），在比较有显著更多细胞凋亡，以控制（左）。与此相反，在珠注射的眼中，其中压力没有上升（右），在TUNEL染色没有显著增加了观察。数据=平均值±SEM。 * = P &lt;0.05; N = 7 - 8（C）的视神经染色的代表性图像，表明来自青光眼（右）的增加甲苯胺蓝累积（黑色箭头）中受损轴突，但不控制眼睛（左）。比例尺= 50微米。这个数字已经被修改福克斯顿等人 ，上午。病理学杂志182（4）:1379年至1390年。

Watch this Scientific Journal Video about Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres at JoVE.com

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

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 ...

experimental-glaucoma-induced-ocular-injection-magnetic

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

17.0K 次观看.  University College London Institute of Ophthalmology. 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.

视频: Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

激光诱导的慢性高眼压表征视觉缺陷的小鼠模型

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

科研

医学

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

神经科学

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

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

A磁条微珠缺血模型，以诱导小鼠高眼压，青光眼相关

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

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 ...

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

Circumlimbal 缝合法诱导大鼠和小鼠青光眼模型的建立

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.

Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.

小鼠中可逆硅油诱发眼部高血压模型

Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

A Hypertonic Saline Injection Technique for Inducing Glaucoma in Rats

Source: Gossman, C. A. et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53831">Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation.</a> J. Vis. Exp. (2016)
This video demonstrates glaucoma induction in an anesthetized rat by injecting hypertonic saline into the episcleral vein. The saline enters the trabecular meshwork via Schlemm&#39;s canal, causing tissue scarring, which blocks aqueous humor outflow and raises intraocular pressure. The elevated pressure damages retinal ganglion cells, leading to glaucoma.

一种高渗盐水注射技术诱导大鼠青光眼

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Epilepsy and Seizure Disorders

Experimental Glaucoma Induction via Iridocorneal Angle Obstruction In a Mouse Model

Source: Bunker, S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52400">Experimental glaucoma induced by ocular injection of magnetic microspheres.</a> J. Vis. Exp. (2015)
In this video, an anesthetized rat undergoes an ocular procedure where magnetic beads are injected into the anterior chamber under a magnetic field to block the iridocorneal angle. This blockage obstructs the trabecular meshwork, preventing aqueous humor drainage and leading to increased intraocular pressure, damaging the retinal ganglion cells and optic nerve. This develops a glaucoma-like condition.

Source: Bunker, S., et al. Experimental glaucoma induced by ocular injection of magnetic microspheres. J. Vis. Exp. (2015)
In this video, an anesthetized ...

Neurodegenerative Diseases

Inducing Experimental Glaucoma Induction via Iridocorneal Angle Obstruction with Magnetic Beads

After anesthetizing the rats, apply ocular ointment to the unoperated contralateral eye to keep it moist during anesthesia. Apply 0.5% pro procaine hydrochloride local anesthetic to the operative eye. Next, wash this eye with 5% povidone iodine in water. After 5 minutes, wick off the povidone iodine and wash the eye with 0.9% sterile saline solution.
Keep the eye moist during anesthesia with regular application of sterile saline. Now, place a toroidal magnet around the eye. To minimize the risk of iris trauma and minimize bead loss, orient the needle tangential to the corneal surface as parallel to the iris as possible. Proceed to inject 25 microliters of 30 milligrams per millimeter gamma irradiated magnetic microspheres into the anterior chamber.
Leave the needle in place for one minute post-injection, to ensure that beads settle into the iridocorneal angle to impede aqueous drainage from the trabecular meshwork. Slightly angle the needle a few seconds after the beads have initially settled to allow some leakage of aqueous, and to minimize transient increases in intraocular pressure.
To ensure continued use of the needle in separate procedures, flush the needle with PBS, then 70% ethanol, followed by distilled water. At this stage, if necessary, remove the magnet and use it to draw beads into areas of incomplete coverage. Leave the magnet in place around the eye for a further 10 minutes post-injection, to ensure beads settle well into the iridocorneal angle.