Name: glutamate and hypoxia as a stress model for the isolated perfused vertebrate retina
Uploaded: 2015-03-22T15:00:00
Description: Watch this Scientific Journal Video about Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina at JoVE.com

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?...

<ol>
	<li>Varma, D. D., Cugati, S., Lee, A. W., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+central+retinal+artery+occlusion:+clinical+presentation+and+management.">A review of central retinal artery occlusion: clinical presentation and management.</a> Eye (Lond). 27, 688-697 (2013).</li><li>Resch, M., Suveges, I., Nemeth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertension-related+eye+disorders).">Hypertension-related eye disorders).</a> Orv Hetil. 154, 1773-1780 (2013).</li><li>Feltgen, N., 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=Multicenter+study+of+the+European+Assessment+Group+for+Lysis+in+the+Eye+(EAGLE)+for+the+treatment+of+central+retinal+artery+occlusion:+design+issues+and+implications.+EAGLE+Study+report+no.+1+:+EAGLE+Study+report+no.+1.">Multicenter study of the European Assessment Group for Lysis in the Eye (EAGLE) for the treatment of central retinal artery occlusion: design issues and implications. EAGLE Study report no. 1 : EAGLE Study report no. 1.</a> Graefes Arch Clin Exp Ophthalmol. 244, 950-956 (2006).</li><li>Dreyer, E. B., Zurakowski, D., Schumer, R. A., Podos, S. M., Lipton, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+glutamate+levels+in+the+vitreous+body+of+humans+and+monkeys+with+glaucoma.">Elevated glutamate levels in the vitreous body of humans and monkeys with glaucoma.</a> Arch Ophthalmol. 114, 299-305 (1996).</li><li>Bertram, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino-acid+levels+in+subretinal+and+vitreous+fluid+of+patients+with+retinal+detachment.">Amino-acid levels in subretinal and vitreous fluid of patients with retinal detachment.</a> Eye (Lond). 22, 582-589 (2008).</li><li>Diederen, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+glutamate+levels+in+the+vitreous+of+patients+with+retinal+detachment).">Increased glutamate levels in the vitreous of patients with retinal detachment).</a> Exp Eye Res. 83, 45-50 (2006).</li><li>Ientile, R., 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=Apoptosis+and+necrosis+occurring+in+excitotoxic+cell+death+in+isolated+chick+embryo+retina.">Apoptosis and necrosis occurring in excitotoxic cell death in isolated chick embryo retina.</a> J Neurochem. 79, 71-78 (2001).</li><li>Mali, R. S., Cheng, M., Chintala, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasminogen+activators+promote+excitotoxicity-induced+retinal+damage.">Plasminogen activators promote excitotoxicity-induced retinal damage.</a> FASEB J. 19, 1280-1289 (2005).</li><li>Ambati, J., 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=Elevated+gamma-aminobutyric+acid,+glutamate,+and+vascular+endothelial+growth+factor+levels+in+the+vitreous+of+patients+with+proliferative+diabetic+retinopathy.">Elevated gamma-aminobutyric acid, glutamate, and vascular endothelial growth factor levels in the vitreous of patients with proliferative diabetic retinopathy.</a> Arch Ophthalmol. 115, 1161-1166 (1997).</li><li>Vorwerk, C. K., 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=Depression+of+retinal+glutamate+transporter+function+leads+to+elevated+intravitreal+glutamate+levels+and+ganglion+cell+death.">Depression of retinal glutamate transporter function leads to elevated intravitreal glutamate levels and ganglion cell death.</a> Invest Ophthalmol Vis Sci. 41, 3615-3621 (2000).</li><li>Schultheiss, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dulbecco's+Modified+Eagle+Medium+is+neuroprotective+when+compared+to+standard+vitrectomy+irrigation+solution.">Dulbecco's Modified Eagle Medium is neuroprotective when compared to standard vitrectomy irrigation solution.</a> Graefes Arch Clin Exp Ophthalmol. 251, 1613-1619 (2013).</li><li>Januschowski, K., 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=Comparing+the+effects+of+two+different+irrigation+solutions+on+an+isolated+perfused+vertebrate+retina.">Comparing the effects of two different irrigation solutions on an isolated perfused vertebrate retina.</a> Ophthalmic Res. 48, 59-66 (2012).</li><li>Sickel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+and+Electrical+Responses+to+Light+Simulation+in+the+Retina+of+the+Frog.">Respiratory and Electrical Responses to Light Simulation in the Retina of the Frog.</a> Science. 148, 648-651 (1965).</li><li>Luke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+perfused+bovine+retina--a+sensitive+tool+for+pharmacological+research+on+retinal+function.+Brain+research.">The isolated perfused bovine retina--a sensitive tool for pharmacological research on retinal function. Brain research.</a> Brain research protocols. 16, 27-36 (2005).</li><li>Henderson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+experimental+data+for+univariate+normality.">Testing experimental data for univariate normality.</a> Clinica chimica acta; international journal of clinical chemistry. 366, 112-129 (2006).</li><li>Alt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuroprotective+potential+of+Rho-kinase+inhibition+in+promoting+cell+survival+and+reducing+reactive+gliosis+in+response+to+hypoxia+in+isolated+bovine+retina.">The neuroprotective potential of Rho-kinase inhibition in promoting cell survival and reducing reactive gliosis in response to hypoxia in isolated bovine retina.</a> Cell Physiol Biochem. 32, 218-234 (2013).</li><li>Green, D. G., Kapousta-Bruneau, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+properties+of+a+new+isolated+rat+retina+preparation.">Electrophysiological properties of a new isolated rat retina preparation.</a> Vision research. 39, 2165-2177 (1999).</li><li>Richter, S. H., Garner, J. P., Wurbel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+standardization:+cure+or+cause+of+poor+reproducibility+in+animal+experiments.">Environmental standardization: cure or cause of poor reproducibility in animal experiments.</a> Nat Methods. 6, 257-261 (2009).</li><li>Dutescu, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifocal+ERG+recordings+under+visual+control+of+the+stimulated+fundus+in+mice.">Multifocal ERG recordings under visual control of the stimulated fundus in mice.</a> Investigative ophthalmology &amp; visual science. 54, 2582-2589 (2013).</li><li>Perlman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+retinal+toxicity+of+drugs+in+animal+models+using+electrophysiological+and+morphological+techniques.">Testing retinal toxicity of drugs in animal models using electrophysiological and morphological techniques.</a> Doc Ophthalmol. 118, 3-28 (2009).</li><li>Mukhopadhyay, A., Gupta, A., Mukherjee, S., Chaudhuri, K., Ray, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Did+myocilin+evolve+from+two+different+primordial+proteins.">Did myocilin evolve from two different primordial proteins.</a> Mol Vis. 8, 271-279 (2002).</li><li>Januschowski, K., 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=Evaluating+retinal+toxicity+of+a+new+heavy+intraocular+dye,+using+a+model+of+perfused+and+isolated+retinal+cultures+of+bovine+and+human+origin.">Evaluating retinal toxicity of a new heavy intraocular dye, using a model of perfused and isolated retinal cultures of bovine and human origin.</a> Graefes Arch Clin Exp Ophthalmol. 250, 1013-1022 (2012).</li><li>Luke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+perfused+bovine+retina--a+sensitive+tool+for+pharmacological+research+on+retinal+function.">The isolated perfused bovine retina--a sensitive tool for pharmacological research on retinal function.</a> Brain Res Brain Res Protoc. 16, 27-36 (2005).</li></ol>

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?...

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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">120 mM NaCl&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,064,041,000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2 mM KCl,&#160;&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,050,010,250</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">0.1 mM MgCl2,&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">58,330,250</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">0.15 mM CaCl2</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td style="width:119px;">111 TA106282</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1.5 mM NaH2PO4/13.5 mM Na2HPO4&#160;&#160;</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">1,065,860,500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">5 mM glucose</td>
			<td style="width:71px;">Merck Pharma, Germany</td>
			<td align="right" style="width:119px;">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?...

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。

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