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 border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:324px;">Name of Material/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:324px;">250-300g female Brown Norway ex-breeder rats</td>
			<td style="width:175px;">Harlan UK</td>
			<td style="width:119px;">203</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Tonolab Rebound Tonometer</td>
			<td style="width:175px;">Tiolat</td>
			<td style="width:119px;">TV02</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:324px;">Ketaset (Ketamine)</td>
			<td style="width:175px;">Fort Dodge Animal health</td>
			<td>BN1000118</td>
			<td>37.5 mg/kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Domitor (medetomidine hydrochloride)</td>
			<td style="width:175px;">Orion Pharma</td>
			<td style="width:119px;">140-999</td>
			<td>0.25 mg/kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Povidone iodine</td>
			<td style="width:175px;">Ecolab</td>
			<td>BN4369LE10</td>
			<td>5% in H2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Minim&#39;s Saline Solution</td>
			<td style="width:175px;">Bausch and Lomb</td>
			<td>PL00033/5017</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Toroidal magnet</td>
			<td style="width:175px;">Supermagnete</td>
			<td>R-10-07-03-N</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Magnetic Microspheres</td>
			<td style="width:175px;">Bangs Laboratories</td>
			<td>UMC4N/9692</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">HBSS</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">14025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">33-guage bevelled needle</td>
			<td style="width:175px;">Hamilton</td>
			<td>7747-01</td>
			<td>Custom needle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Luer tip syringe</td>
			<td style="width:175px;">Hamilton</td>
			<td>80601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Antisedan (atipemezole hydrochloride )</td>
			<td style="width:175px;">Orion Pharma</td>
			<td style="width:119px;">141-003</td>
			<td>0.25 mg/kg</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Chloramphenicol ointment</td>
			<td style="width:175px;">Medicom</td>
			<td>18956-0005</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">TUNEL staining kit</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">G3250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Triton X-100</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">DAPI</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:324px;">Vectashield Mounting Media</td>
			<td style="width:175px;">Vector Labs</td>
			<td style="width:119px;">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

Research

JoVE Journal

Medicine

16.9K 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

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

生物医学应用海藻酸钠微球的一代

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

科研

医学

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

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 ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

心脏异常的实验性自身免疫性心肌炎的无创性评估磁共振成像显微镜在鼠标

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

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 ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

脉络细胞自体移植治疗老年性黄斑变性的再生疗法: 初步体内报告

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits. Thanks to animal models, basic research can investigate the mechanisms underlying pathological processes such as MetS. Here, we describe the methods used to develop an experimental rabbit model of diet-induced MetS and its assessment. After a period of acclimation, animals are fed a high-fat (10% hydrogenated coconut oil and 5% lard), high-sucrose (15% sucrose dissolved in water) diet for 28 weeks. During this period, several experimental procedures were performed to evaluate the different components of MetS: morphological and blood pressure measurements, glucose tolerance determination, and the analysis of several plasma markers. At the end of the experimental period, animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia with low HDL, high LDL, and an increase of triglyceride (TG) levels, thus reproducing the main components of human MetS. This chronic model allows new perspectives for understanding the underlying mechanisms in the progression of the disease, the detection of preclinical and clinical markers that allow the identification of patients at risk, or even the testing of new therapeutic approaches for the treatment of this complex pathology.

We describe methods to develop an experimental model of diet-induced metabolic syndrome (MetS) in rabbits using a high-fat, high-sucrose diet. Animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia, thus reproducing the main components of human MetS. This chronic model will allow acquisition of knowledge underlying mechanisms of disease progression.

家兔膳食诱导代谢综合征的实验模型: 方法论考虑、发展和评价

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased ...

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 Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer&#39;s tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

由康卡纳林注射到乳原区引起的水性干眼病的兔子模型：药物功效研究的应用

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for ...