This paper is dedicated to my beloved wife Maren and our little Karl.

1.准备牛眼睛<ol><li>获得牛眼中的动物屠宰后直接。 </li><li>输送保护眼睛"Sickel溶液"含有120mM氯化钠，2mM的氯化钾，0.1mM的MgCl 2的，0.15毫氯化钙 ，1.5mM的的NaH 2 PO 4，13.5毫的Na 2 HPO 4和5mM葡萄糖在一个特殊的介质RT。 </li><li>用昏暗红光暗适应的条件下进行视网膜的制备。 </li><li>取出眼睛的前部。执行赤道切口长约4毫米后的角膜缘。此后在一块除去角膜，虹膜，睫状体和透镜。请Sickel-解决方案的视网膜。 </li><li>机械地松开玻璃体附着物视网膜表面，并从打开的眼杯除去玻璃体。 </li><li>随后划分眼睛成四个象限和切出圆约领域直径7mm使用环钻。 </li><li>轻轻分开视网膜从色素上皮，并将其放置在记录设备上的内避光的盒子。该记录装置由一个塑料维护者在中间的网格的;放置在网格的视网膜，然后用一个塑料环，直接在电极上固定。 注:塑料维护者有两个通道，以允许介质的恒定流速。 </li></ol> 2.录音电图（ERG） <ol><li>为了记录的电图，使用两个银/氯化银电极在视网膜上的任一侧和灌注的视网膜在大约恒定的灌注速度1毫升/分钟和37℃的恒温。使用与氧饱和"Sickel-的解决方案"。 </li><li>开始测量之前，暗适应视网膜（在所有测量保护其不受光），并使用了5分钟的刺激间隔。使用1赫兹单一白色氙气闪光灯的刺激强度设定为6.3 MLX在视网膜表面。 </LI&gt; <li>使用校准的中性密度滤光片和10微秒的光刺激由计时器为了具有最佳的反应进行控制。 </li><li>测量和处理数据，过滤的ERG，并用草RPS312RM放大器放大它（100赫兹的高通滤波器，50赫兹的陷波滤波器，100,000×放大）。试图筛选出可能的干扰频率，可能会干扰信号。为了处理数据，使用模拟 - 数字数据采集板的台式计算机（PC兼容）上。 </li><li>在恒定灌注的暗适应期后，测量的电信号的幅度，直到稳定b波振幅被记录。 注:振幅被认为是稳定的，如果五个单测量达到一个平均值和偏差小于10％。单次测量的一个很好的例子是在图1。 </li><li>开始测试，无论是纯氮气代替纯氧（单实验数n = 5）来测试缺氧或250微米谷氨酸（N = 5）。 </li><li>记录电反应，每5分钟45分钟。 </li><li>在测试期结束后，灌注的视网膜与氧饱和75分钟的标准培养基，并期待在b-波振幅的变化。这是洗出阶段。测量从一个波到b-波的峰值的波谷的b-波幅度。 </li><li>调查低氧或谷氨酸上暗的条件下的感光体电位的效果，抑制b-波通过加入1mM至所述营养液。 </li><li>记录一个稳定的光感受器电位30分钟后，执行程序和以前一样，露出了视网膜45分钟，以不同的冲洗液以1 mM的天门冬氨酸。使用相同的洗脱期（步骤2.8）正如前面提到的。 </li></ol> 3.数据分析<ol><li>为了统计评估数据，确保正常的分布所有的数据， 如使用柯尔莫哥洛夫-斯米尔诺夫测试15。 </li><li>计算的a-和b-波振幅的百分比在曝光阶段之后的减少相比，最后一次测量的论述之前。 45分钟后，比较ERG-振幅的减小 - 在暴露时段的结束 - 到ERG应用之前测量。 </li><li>比较在冲洗阶段相应幅度的论述之前结束的A股和B超波，研究可能的复苏。 </li><li>进行统计分析，使用该软件JMP统计软件SPSS或软件。整个计算的平均值±标准差的数据。估计由相应的统计检验的意义。 注:这些测试可能会根据不同的实验范围。在这种背景下，使用学生的配对t检验。 </li></ol>

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The authors have nothing to disclose.

在这项研究中，在缺氧的45分钟后的b波振幅的显著影响被发现。本次减持仍是冲洗阶段后显著。可以观察到在感光体电位类似的效果。 结果被其他公布的数据16，给我们研究缺氧后可能神经保护作用的机会的支持。 后45分钟的论述250μM谷氨酸，我们没有发现只在b-波振幅为完全可逆的清除期的结束在统计学显著影响。感光体电位不是由250μM谷氨酸的影响。只有内视网膜受缺氧的事实表明该变化是非常微妙的，我们，因此，有可能的神经保护一个非常敏感的和标准化的指示器。天冬氨酸特异性抑制传输从感光体向地平线人或双极细胞，从而抑制b-波。 这一发现是违背绿色DG和Kapousta -布诺NV，谁发现，250和500毫米谷氨酸浓度比单独17家媒体的B波记录是随着时间的推移更加稳定的结果。为了解释这个矛盾，在设置几个不同需要加以考虑:Sickel-溶液用于在该模型中使用的其它出版物林格溶液。 测量进行与数组中的其他出版物有一个电极。而其他出版物所描述的新鲜大鼠的眼睛牛眼从屠宰场被使用。其效果是可逆的，在高浓度的谷氨酸一个时间的抑制。众所周知，一个特定的谷氨酸的量非常低的是必要的良好的维护眼睛和更高的量是有毒的。我们可以假设，在牛的眼睛从SLaughterhouse更多相比，导致谷氨酸含量是不同的实验添加谷氨酸比预期新鲜烹制的眼睛谷氨酸被释放。 测量使用阵列在整个视网膜而不是一个电极，其主要受最接近细胞的反应，也可能导致更长的存活时间，由于较低的机械损伤。最后，该媒体的组合物是至关重要的。在20世纪60年代，Sickel教授投入巨资开发这个 ​​专业媒体对这些测量，他能够找到一个优化的媒体，而无需为谷氨酸稳定的视网膜反应13,14。 所描述的模型依赖于一个孤立的视网膜上，而不是整个动物。 在体内系统中，眼睛是由从该动物的其他器官的血液-视网膜屏障隔离。该模型的优点是，在干扰研究动物的参数，如麻醉或电极的位置不发生，其允许更高程度的标准化。标准化是在动物实验中的关键点上，特别是关于眼18。 该方法的局限性是短期测试期间和事实，即它不是一个动物模型。我们从经验中知道，ERG幅度保持稳定，约8小时，因此在介绍中提到的测试期可以延长到更长的论述和随访期，但每个人都应该考虑的不再是单一的实验延长，更可能会出现更高的标准偏差。 该模型的一个缺点是，因此，没有长期的研究可以进行和操作，以在视网膜只有有限数量是可能的。为广泛的操作和特殊尔格等作为在Dutescu 等人所述通过SLO引导多焦点ERG。例如， 在体内实验s为必要的19。 这种模式可以很容易地用于例如从摘除眼睛人类视网膜外植。由于这个敏感材料复杂获得，对牛视网膜实验是比较可行的，但应理解牢记这一点。 在协议中的关键步骤是运输和暗适应条件下准备视网膜。这是很重要的，以获得最大可能的响应。它有时可能需要几分钟从底层色素上皮轻轻分开视网膜。的冲压出视网膜轻轻摇动有时有助于这一过程。同时将在视网膜上的网，它放置在视网膜上的净的外视网膜是重要的。 trephanization后，在视网膜的边缘将稍微弯曲向上指示的内视网膜层的位置。 牛视网膜更类似于人的视网膜比较d，来啮齿动物眼睛的，因为类似的玻璃体透镜比和血管结构类似于人眼20的1视网膜;因此，虽然小型实验室动物如大鼠或小鼠被广泛用于测试视网膜的生物相容性，对这样的动物进行的毒性研究往往不太适用于人类20。所述myocilin基因的系统发育分析-一种基因在其突变形式引起的常染色体显性少年开角型青光眼-表明牛基因更密切相关的人基因比大鼠或小鼠21。 Myocilin可以发现在小梁细胞，以及在视网膜上。最后我们发现牛和人的孤立和尔格ERG效果12之间的良好关系。 在我们的模型天冬氨酸受雇于废除b波揭露感光潜在的P III。这给研究者的机会，以区分之间的内视网膜网络和光感受器功能22上的影响。而b波是一个关于集成功能更加敏感参数，所述一个波振幅更稳定，应力更耐，由于这样的事实，该一个波仅反映了光感受器的光反应它。与此相反，b波取决于不同的视网膜细胞的细胞 - 细胞相互作用和光感受器的活性。通过使用天冬氨酸以分离一个波是可能的检查操作的独自23的感光体的作用。 在这项研究中，可以用来测试神经保护剂的标准化的神经毒性模型中评估。作为一个前景将是有趣的，相关电生理功能与蛋白的生化分析或关联与细胞代谢，甚至表达的功能。

神经保护一直在调查研究的眼科在过去几十年的强场。视网膜是氧合显著取决于并强烈地受到其周围细胞的代谢的影响高度敏感的神经元网络。涉及神经细胞的损伤大眼病症是视网膜血管闭塞，青光眼和视网膜脱离。 视网膜动脉闭塞，作为网膜血管闭塞症，例如，导致视力突然损失，由于视网膜内层1缺氧。它通常与一般的血管病理学2和导线的持久视觉损失相关联，只有8％的患者恢复视力显著1。虽然动脉纤溶 ​​已建议作为一种治疗选择，好处不能显示在一个随机临床试验3。 青光眼和视网膜脱离都有增加谷氨酸浓度4-6。生理条件下，谷氨酸遇到作为兴奋性发射机在整个中枢神经系统和视网膜内层7,8。提高谷氨酸含量已经发现不仅在青光眼和视网膜脱离5,6而且在增殖性糖尿病视网膜病变9。谷氨酸的增加可能会导致兴奋性毒性，因此，神经细胞的损伤10。在视网膜脱离和在某些情况下在视网膜上（玻璃体）增生性糖尿病性视网膜病的手术大多数情况下是必要的。在玻璃体机械操纵，明亮的光线光纤或在长期的经营中灌溉解决方案的高流速产生的剪切应力对视网膜11,12施加了额外的压力。 所有的提到的疾病的共同之处在于所述病理定位于雷廷一个单独构成的眼科界的挑战，想方设法保护视网膜神经上一个系统。 电图（ERG）是用于在体内光感受器功能（一个波）和内视网膜（b波）的功能的评价的标准方法。 ERG的被引入到角膜银电极来测量和眼睛正在刺激光的增加的水平，以检测在杆或视锥细胞或视网膜内层缺陷。在视网膜的不同的缺陷可以通过改变幅度（响应的强度）或ERG的延迟（时间到响应的时间间隔）来检测。不同的ERG协议和测量方法（模式-ERG，多焦-ERG或明场ERG）允许缺陷进一步分化。分离的视网膜的技术最近已经引入，使得可以评估，而不脱离如一个研究动物的干扰在视网膜上的影响一般反应13,14。 正是这种研究，评估和引进定义和标准化应力模型的缺氧和谷氨酸应力的隔离灌流视网膜的目的。因此，我们希望能为今后的某些代理商或眼内冲洗液的保护作用的研究奠定了基础。

<table><tbody><tr><td>120 mM NaCl&amp;nbsp;</td><td>Merck Pharma, Germany</td><td>1,064,041,000</td><td></td></tr><tr><td>2 mM KCl,&amp;nbsp;&amp;nbsp;</td><td>Merck Pharma, Germany</td><td>1,050,010,250</td><td></td></tr><tr><td>0.1 mM MgCl2,&amp;nbsp;</td><td>Merck Pharma, Germany</td><td>58,330,250</td><td></td></tr><tr><td>0.15 mM CaCl2</td><td>Merck Pharma, Germany</td><td>111 TA106282</td><td></td></tr><tr><td>1.5 mM NaH2PO4/13.5 mM Na2HPO4&amp;nbsp;&amp;nbsp;</td><td>Merck Pharma, Germany</td><td>1,065,860,500</td><td></td></tr><tr><td>5 mM glucose</td><td>Merck Pharma, Germany</td><td>40,741,000</td><td></td></tr></tbody></table>

glutamate and hypoxia as a stress model for the isolated perfused vertebrate retina

神经保护已调查的一个强电场在眼科研究在过去的几十年，并影响疾病如青光眼，视网膜血管阻塞症，视网膜脱落，以及糖尿病视网膜病变。它是本研究引入一个标准化应力模型为未来的临床前治疗试验的对象。 制备牛视网膜和灌注的饱和氧标准溶液中，ERG记录。记录稳定的B-波，缺氧（纯的N 2）或谷氨酸应力（250微米谷氨酸）后施加45分钟。调查于光感受器功能单独的影响，1毫天冬氨酸加到得到波。 ERG恢复75分钟进行监测。 对于缺氧，在为87.0％，波幅下降注意到（P &lt;0.01），后45分钟（减少36.5％的冲刷P = 0.03结束后）博览会时间。此外，初始DECR缓解的记录87.23％b波振幅，即达到统计学意义（p &lt;0.01，减少了25.5％，在冲洗结束时，p值= 0.03）。 为250微米的谷氨酸，初始还原，随后还原了1.9％（P&gt; 0.05）a-波振幅（P&gt; 0.05）7.8％。减少b波振幅（P &lt;0.01），指出了83.7％;减少了75分钟的冲洗后为2.3％（P = 0.62）。在这项研究中，一个标准化的应力模型被呈现，可能是有用的，以确定在未来可能的神经保护作用。

1小时视网膜制剂与氧饱和标准溶液（ 图1A和B）的灌注后ERG-振幅表现出稳定和单次测量之间的振幅的变化较小。 pH值，渗透压，温度和PO 2（除了低氧测试）保持恒定为所有测试。 为了隔离从内视网膜的信号的感光信号，1mM的天冬氨酸加入到标准溶液来抑制b-波（ 图1A）。在缺氧的效果测试，为87.0％，波幅下降注意到（P &lt;0.01），后45分钟的曝光时间。在冲洗结束后，下降了36.5％，有人指出，有统计学显著（P = 0.03， 图2A）。此外，在87.23％，b波振幅初步录得跌幅，这同样达到统计学意义（P &lt;0.01）。在这种设置减少了25.5％有人指出，这是统计上显著（p值= 0.03， 图2B）。 论述与250微米谷氨酸，7.8％的非显著减少（P&gt; 0.05）所定义的时间间隔后检测幅度的波数据。其次是非显著减少1.9％（P&gt; 0.05， 图3A）。单次测量示于表1和2。 关于b波，降低ERG的幅度由83.7％录得的统计学显著（P &lt;0.01， 图3B）。在冲洗结束时，一个b波恢复指出导致75分钟灌注用标准溶液（p值= 0.62）后的非显著减少为2.3％。 <img alt="图1" src="/files/ftp_upload/52270/52270fig1highres.jpg" /> 图1:从离体牛视 ​​网膜的ERG测量实例 （A）的一个波如图4所示。离体灌注牛视网膜的ERG。 b-波通过加入1mM的天冬氨酸到营养液抑制。（B）中的b波是暗光条件下占主导地位。 10毫秒的光刺激在6.3毫升×光强度使用。 <img alt="图2" src="/files/ftp_upload/52270/52270fig2highres.jpg" /> 图2:效果缺氧的45分钟中的（A）中的曝光时间之后的波和在ERG的（B）的 b波的振幅。代表药物系列平均值（N = 5）。曲线上方单杠标志着缺氧时间。虚线（A）标志着天门冬氨酸1毫米的应用揭露感光潜力。标准偏差为每个系列实验直接之前和之后，以及在试验结束时给出。在使用标准偏差的时间点进行统计分析（直接之前和之后应用索引离子以及在试验结束时）:（A）在波的87.0％，幅度注意到后相比，试验初期45分钟博览会时间的减少。在试验结束时，显著下降36.5％，有人指出，（B）的87.23％，b波幅度的显著录得跌幅在博览会结束时间。在试验结束时，显著减少25.5％，有人指出。 <img alt="图3" src="/files/ftp_upload/52270/52270fig3highres.jpg" /> 图3:影响施加45分钟的（A）的 250μM谷氨酸一波振幅与离体灌注牛视网膜平均的代表性药物系列的ERG的（B）的 b波振幅（N = 5） 。曲线上方的水平杆马克谷氨酸的应用。虚线（A）标志着天门冬氨酸1毫米的应用揭露感光潜力。站ndard偏差每个系列实验直接之前和之后，以及在试验结束时给出。 （A）的250微米谷氨酸非显著减少一个波的曝光时间后:在时间点与标准偏差（直接前和使用后在试验结束以及）进行统计分析幅度（7.8％）进行检测。在试验结束时，发现非显著减少1.9％。（B）关于b波，显著降低ERG的幅度由83.7％的记录。在试验结束时，非显著减少2.3％，有人指出。 <table border="0"><tbody><tr><td> 时间[分钟] </td><td> b波振幅[μV] </td><td> SD </td><td> 一个波振幅[μV] </td><td> SD </td></tr><tr><td align="right"> 0 </td><td align="right"> 9.2 </td> &lt;TD ALIGN ="右"&gt; 0.836660027 </td><td align="right"> -10.4 </td><td align="right"> 1.140175425 </td></tr><tr><td align="right">五</td><td align="right"> 9.2 </td><td align="right"> 1.095445115 </td><td align="right"> -10 </td><td align="right"> 1 </td></tr><tr><td align="right"> 10 </td><td align="right"> 10 </td><td align="right"> 0 </td><td align="right"> -10.6 </td><td align="right"> 0.547722558 </td></tr><tr><td align="right"> 15 </td><td align="right"> 9.4 </td><td align="right"> 1.140175425 </td><td align="right"> -9.6 </td><td align="right"> 0.547722558 </td></tr><tr><td align="right"> 20 </td><td align="right"> 9.4 </td><td align="right"> 0.547722558 </td><td align="right"> -10 </td><td align="right"> 1 </td></tr><tr><td align="right"> 25 </td><td align="right"> 9.4 </td><td align="right"> 0.894427191 </td><td align="right"> -10.8 </td><td align="right"> 0.447213595 </td></tr><tr><td a河旁="右"&gt; 30 </td><td align="right"> 4.2 </td><td align="right"> 2.774887385 </td><td align="right"> -6.6 </td><td align="right"> 2.50998008 </td></tr><tr><td align="right"> 35 </td><td align="right"> 4 </td><td align="right"> 2.449489743 </td><td align="right"> -5 </td><td align="right"> 2.236067977 </td></tr><tr><td align="right"> 40 </td><td align="right"> 3.8 </td><td align="right"> 1.788854382 </td><td align="right"> -5 </td><td align="right"> 1 </td></tr><tr><td align="right"> 45 </td><td align="right"> 3.6 </td><td align="right"> 1.341640786 </td><td align="right"> -5 </td><td align="right"> 2.121320344 </td></tr><tr><td align="right"> 50 </td><td align="right"> 2.4 </td><td align="right"> 1.140175425 </td><td align="right"> -4 </td><td align="right"> 1.870828693 </td></tr><tr><td align="right"> 55 </td><td align="right"> 2.2 </td><td align="right"> 0.836660027 </td><td align="right"> -3.4 </TD&gt; <td align="right"> 1.140175425 </td></tr><tr><td align="right"> 60 </td><td align="right"> 2.6 </td><td align="right"> 0.547722558 </td><td align="right"> -3.4 </td><td align="right"> 1.816590212 </td></tr><tr><td align="right"> 65 </td><td align="right"> 1.8 </td><td align="right"> 0.836660027 </td><td align="right"> -2.8 </td><td align="right"> 1.303840481 </td></tr><tr><td align="right"> 70 </td><td align="right"> 1.2 </td><td align="right"> 0.447213595 </td><td align="right"> -1.4 </td><td align="right"> 0.894427191 </td></tr><tr><td align="right"> 75 </td><td align="right"> 4.2 </td><td align="right"> 0.836660027 </td><td align="right"> -5.2 </td><td align="right"> 2.683281573 </td></tr><tr><td align="right"> 80 </td><td align="right"> 5.8 </td><td align="right"> 1.303840481 </td><td align="right"> -6 </td><td align="right"> 2.828427125 </td></tr><tr><td align="right"> 85 </td><td aligN ="右"&gt; 6.2 </td><td align="right"> 1.095445115 </td><td align="right"> -4.8 </td><td align="right"> 0.836660027 </td></tr><tr><td align="right"> 90 </td><td align="right"> 6.8 </td><td align="right"> 1.095445115 </td><td align="right"> -6.2 </td><td align="right"> 2.387467277 </td></tr><tr><td align="right"> 95 </td><td align="right"> 7.4 </td><td align="right"> 1.816590212 </td><td align="right"> -5.6 </td><td align="right"> 2.302172887 </td></tr><tr><td align="right"> 100 </td><td align="right"> 6.4 </td><td align="right"> 0.547722558 </td><td align="right"> -6.2 </td><td align="right"> 2.863564213 </td></tr><tr><td align="right"> 105 </td><td align="right"> 6.6 </td><td align="right"> 0.894427191 </td><td align="right"> -7.2 </td><td align="right"> 2.049390153 </td></tr><tr><td align="right"> 110 </td><td align="right"> 6.2 </td><td align="right"> 1.643167673 </td><td align="right"> -6.4 </td> &lt;TD ALIGN ="右"&gt; 2.966479395 </td></tr><tr><td align="right"> 115 </td><td align="right"> 8.8 </td><td align="right"> 3.898717738 </td><td align="right"> -6 </td><td align="right"> 3.16227766 </td></tr><tr><td align="right"> 120 </td><td align="right"> 7.4 </td><td align="right"> 1.516575089 </td><td align="right"> -6 </td><td align="right"> 2.34520788 </td></tr><tr><td align="right"> 125 </td><td align="right"> 6.8 </td><td align="right"> 1.095445115 </td><td align="right"> -6 </td><td align="right"> 2.645751311 </td></tr><tr><td align="right"> 130 </td><td align="right"> 7 </td><td align="right"> 1 </td><td align="right"> -6.6 </td><td align="right"> 1.816590212 </td></tr></tbody></table> 表1:柯尔莫哥洛夫-斯米尔诺夫的结果。 <table border="0"><tbody><tr><td>时间[分钟] </td><td> b波振幅[μV] </td><td> SD </td><td>一个波振幅[μV] </td&gt; <td> SD </td></tr><tr><td align="right"> 0 </td><td align="right"> 9.25 </td><td align="right"> 0.5 </td><td align="right"> -10 </td><td align="right"> 1 </td></tr><tr><td align="right">五</td><td align="right"> 10.5 </td><td align="right"> 0.577350269 </td><td align="right"> -10 </td><td align="right"> 1 </td></tr><tr><td align="right"> 10 </td><td align="right"> 10.25 </td><td align="right"> 0.5 </td><td align="right"> -10.4 </td><td align="right"> 0.894427191 </td></tr><tr><td align="right"> 15 </td><td align="right"> 10.5 </td><td align="right"> 0.577350269 </td><td align="right"> -9.6 </td><td align="right"> 0.894427191 </td></tr><tr><td align="right"> 20 </td><td align="right"> 10 </td><td align="right"> 0.816496581 </td><td align="right"> -9.6 </td><td align="right"> 0.547722558 </td></tr><tr><td align="right"> 25 </td><td align="right"> 10.75 </td><td align="right"> 0.5 </td><td对齐="正确"&gt; - 10.2 </td><td align="right"> 0.836660027 </td></tr><tr><td align="right">三十</td><td align="right"> 3 </td><td align="right"> 1.825741858 </td><td align="right"> -8.4 </td><td align="right"> 2.701851217 </td></tr><tr><td align="right"> 35 </td><td align="right"> 6 </td><td align="right"> 3.559026084 </td><td align="right"> -9 </td><td align="right"> 1 </td></tr><tr><td align="right"> 40 </td><td align="right"> 5.25 </td><td align="right"> 2.62995564 </td><td align="right"> -8.4 </td><td align="right"> 2.966479395 </td></tr><tr><td align="right"> 45 </td><td align="right"> 2.75 </td><td align="right"> 2.061552813 </td><td align="right"> -9 </td><td align="right"> 1.414213562 </td></tr><tr><td align="right"> 50 </td><td align="right"> 2.75 </td><td align="right"> 0.957427108 </td><td align="right"> -8 </td><td align="right"> 0.707106781 </td></tr><tr><td align="right"> 55 </td><tdALIGN ="右"&gt; 2.5 </td><td align="right"> 1 </td><td align="right"> -10.2 </td><td align="right"> 1.095445115 </td></tr><tr><td align="right"> 60 </td><td align="right"> 2.25 </td><td align="right"> 0.957427108 </td><td align="right"> -8.4 </td><td align="right"> 1.816590212 </td></tr><tr><td align="right"> 65 </td><td align="right"> 2 </td><td align="right"> 1.414213562 </td><td align="right"> -8.6 </td><td align="right"> 1.140175425 </td></tr><tr><td align="right"> 70 </td><td align="right"> 1.75 </td><td align="right"> 0.5 </td><td align="right"> -9.4 </td><td align="right"> 2.701851217 </td></tr><tr><td align="right"> 75 </td><td align="right"> 8 </td><td align="right"> 3.464101615 </td><td align="right"> -9.2 </td><td align="right"> 2.28035085 </td></tr><tr><td align="right"> 80 </td><td align="right"> 9.5 </td><td align="right"> 3.696845502 </td><td align="right"> -9 </td><td align="right"> 1 </TD&gt; </tr><tr><td align="right"> 85 </td><td align="right"> 8 </td><td align="right"> 2.160246899 </td><td align="right"> -10.8 </td><td align="right"> 2.48997992 </td></tr><tr><td align="right"> 90 </td><td align="right"> 8.25 </td><td align="right"> 0.957427108 </td><td align="right"> -9.2 </td><td align="right"> 2.167948339 </td></tr><tr><td align="right"> 95 </td><td align="right"> 9.25 </td><td align="right"> 2.872281323 </td><td align="right"> -10 </td><td align="right"> 1.870828693 </td></tr><tr><td align="right"> 100 </td><td align="right"> 9.75 </td><td align="right"> 0.957427108 </td><td align="right"> -9.6 </td><td align="right"> 1.140175425 </td></tr><tr><td align="right"> 105 </td><td align="right"> 13.5 </td><td align="right"> 3.872983346 </td><td align="right"> -9.2 </td><td align="right"> 1.643167673 </td></tr><tr><td align="right"> 110 </td><td align="right"> 11.75 </td><td aligN ="右"&gt; 2.5 </td><td align="right"> -9.6 </td><td align="right"> 1.140175425 </td></tr><tr><td align="right"> 115 </td><td align="right"> 9 </td><td align="right"> 1.825741858 </td><td align="right"> -10 </td><td align="right"> 1.870828693 </td></tr><tr><td align="right"> 120 </td><td align="right"> 11 </td><td align="right"> 3.366501646 </td><td align="right"> -9.8 </td><td align="right"> 1.303840481 </td></tr><tr><td align="right"> 125 </td><td align="right"> 11 </td><td align="right"> 2.708012802 </td><td align="right"> -10.8 </td><td align="right"> 0.447213595 </td></tr><tr><td align="right"> 130 </td><td align="right"> 10.5 </td><td align="right"> 0.577350269 </td><td align="right"> -10 </td><td align="right"> 1 </td></tr></tbody></table> 表2:对缺氧b波振幅和波振幅代表测量。 <table border="0"><tbody><tR&gt; <td></td><td>谷氨酸一个波数据</td><td>谷氨酸b波数据</td><td>缺氧波数据</td><td>缺氧b波数据</td></tr><tr><td> ð </td><td> 0.103827009 </td><td> 0.17415038 </td><td> 0.195 </td><td> 0.125 </td></tr><tr><td> p值</td><td> 0.928603977 </td><td> 0.375501627 </td><td> 0.250 </td><td> 0.781 </td></tr><tr><td>阿尔法</td><td> 0.05 </td><td> 0.05 </td><td> 0.05 </td><td> 0.05 </td></tr></tbody></table> 表3:250μM谷氨酸b波振幅代表测量和波振幅。

Watch this Scientific Journal Video about Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina at JoVE.com

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

有了这个研究中，我们实行规范的应力模型的隔离灌流牛视网膜对未来临床治疗试验。要么缺氧（纯的N 2）或谷氨酸应力（250μM谷氨酸）对由a-和b-波振幅为代表视网膜功能的效果进行了评价。

谷氨酸和缺氧作为离体脊椎动物视网膜应力模型

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

glutamate-hypoxia-as-stress-model-for-isolated-perfused-vertebrate

Nanyang Technological University

Research

JoVE Journal

Medicine

7.8K 次观看.  University Eye Hospital Tübingen. 有了这个研究中，我们实行规范的应力模型的隔离灌流牛视网膜对未来临床治疗试验。要么缺氧（纯的N 2）或谷氨酸应力（250μM谷氨酸）对由a-和b-波振幅为代表视网膜功能的效果进行了评价。 

视频: Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

科研

神经科学

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

缺氧缺糖再给氧作为<em&gt;在体外</em&gt;缺血再灌注损伤模型的研究血脑屏障功能障碍

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

医学

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

大鼠视网膜仿生化学调节的神经递质谷氨酸的体外实验方法

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

生物工程

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 ...

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

由瞬态双边常见胡萝卜动脉闭塞引起的缺血性视网膜损伤的 Murine 模型

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离 体 模型

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

谷氨酸和缺氧作为离体脊椎动物视网膜应力模型

摘要

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此视频中的章节

血管再生在中枢神经系统中使用的小鼠视网膜评估

神经血管重塑的分析Entorhino，海马脑切片培养

缺氧缺糖再给氧作为

大鼠视网膜仿生化学调节的神经递质谷氨酸的体外实验方法

青光眼诱导在程序

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

由瞬态双边常见胡萝卜动脉闭塞引起的缺血性视网膜损伤的 Murine 模型

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离体模型

谷氨酸和缺氧作为离体脊椎动物视网膜应力模型

摘要

探索更多视频

此视频中的章节

血管再生在中枢神经系统中使用的小鼠视网膜评估

神经血管重塑的分析Entorhino，海马脑切片培养

缺氧缺糖再给氧作为

大鼠视网膜仿生化学调节的神经递质谷氨酸的体外实验方法

青光眼诱导在程序

在缺血样条件下监测星形胶质细胞的反应性和增殖体外

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

由瞬态双边常见胡萝卜动脉闭塞引起的缺血性视网膜损伤的 Murine 模型

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离 体 模型

成年小鼠视网膜的视网膜外植体作为研究视网膜神经血管疾病的离体模型