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Neuroscienze

受体放射自显影协议血管紧张素Ⅱ受体的本地化的可视化

Published: June 07, 2016
doi:
10.3791/53866
, , ,
1Farquhar College of Arts and Sciences,Nova Southeastern University, 2Department of Pharmaceutical Sciences, College of Pharmacy,Nova Southeastern University, 3School of Medicine,University of Colorado

Summary

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

Abstract

这个协议描述了使用特异于血管紧张素II受体进行受体放射自显影映射放射性为血管紧张素Ⅱ（血管紧张素II）中的大鼠脑受体结合模式。

组织标本收获并保存在-80℃。低温恒温器是用来冠部分组织（脑）和部分解冻 – 安装到带电的幻灯片。制作在载玻片上的组织切片在125 I SI-血管紧张素II孵育放射性标记血管紧张素Ⅱ受体。相邻的幻灯片被分成两组：“非特异性结合”非放射性标记的血管紧张素II的受体饱和的浓度，或存在（NSP）的AT 1血管紧张素II受体亚型（AT 1R）选择性血管紧张素II受体拮抗剂，与“总结合'没有AT 1 R拮抗剂。 AT 2血管紧张素Ⅱ受体亚型（AT 2 R）饱和浓度的拮抗剂（PD123319，10μM）也出现在incubati关于缓冲区限制125 I-SI-血管紧张素II结合到AT 1 R亚型。在30分钟预温育在约22℃，NSP载玻片暴露于10μM的PD123319和氯沙坦，而'总结合'载玻片暴露于10μM的PD123319。载玻片然后用125 I SI-血管紧张素II在缓冲液中培养在PD123319存在关于'总结合'和PD123319和氯沙坦为NSP在测定缓冲液，随后几个“洗涤”，和水以除去盐和非特异性结合放射性。幻灯片使用吹塑机，然后暴露于放射自显影胶片使用一个专门的电影和录像带干燥。胶片显影和图像被扫描成用于使用专有成像系统和一个电子表格视觉和定量密度的计算机。另外一组幻灯片的硫堇染色进行组织学的比较。

使用受体放射自显影的优点是可视化的能力血管紧张素II受体原位 ，一个组织试样的一个区段内，和解剖学通过比较相邻的组织学参考部分确定的组织的区域。

Introduction

心血管疾病仍然是死亡和残疾在美国，造成人员死亡，在美国超过30％的主要原因在2011年1，从美国心脏协会最近的统计数据表明，超过三分之一的人有一个或更多类型的心血管疾病。心血管研究继续迈大步对了解这种疾病，但作为一代开始变老当务之急是要继续努力。肾素-血管紧张素系统（RAS）的主要由促进动脉粥样硬化，炎症，全身性的血管收缩，和激活交感神经系统的（ 图1）2-8起着在心血管系统的调节中发挥中心作用。

在RAS是当肾的肾小球旁细胞分泌肾素进入血液响应于血压下降时启动的荷尔蒙系统，增加sympathetic刺激，或由致密斑流钠降低。肾素代谢血管紧张素原（在肝脏中合成），以形成血管紧张素I（血管紧张素Ⅰ）。血管紧张素I被随后通过血管紧张素转换酶（ACE）代谢，一种外酶对血管内皮细胞的腔侧，主要在肺，肾，以形成血管紧张素II（ANG II）中，在RAS的主要效应的肽。血管紧张素Ⅱ是能够激活两种接受亚型;的1型受体（AT 1 R）和2型受体（AT 2 R），两者调节心血管系统，保持体液和电解质平衡和现在被认为影响认知功能和神经变性疾病进程8,9。本地，特定脑-RAS报道独立合成血管紧张素II。在大脑中，前体蛋白血管紧张肽原在由肾素样酶3转化为血管紧张素我星形胶质细胞10合成，可能是肾素原结合于肾素原受体11，并且随后通过其在大脑中的神经元12的胞表面上大量表达血管紧张素转换酶转化成血管紧张素II。产生的血管紧张素II这intrabrain是对大脑的AT 1和AT个从血源性血管紧张素Ⅱ分离2受体的激动剂。

而对AT 1 R起着重要的生理作用，这是更好地了解整个身体的病理生理作用，主要影响心血管系统和肾脏（ 图2）。当血管紧张素II结合到AT 1 R，它会导致血管收缩;增加血流阻力和血压升高。它还促进醛固酮和加压素的合成和分泌，从而增加钠和水潴留。这些效果也可以引起缺血性脑损伤和认知障碍，并链接到帕金森氏病，阿尔茨海默氏病，和diabeTES，以及最近被鉴定为影响学习和记忆13-15。有在RAS在于一个反馈回路的AT在肾脏肾小球旁细胞1 R抑制肾素分泌。有趣的是，AT 2 R一般反调控的AT 1 R，导致在许多其他16-20血管扩张，轴突生长，轴突再生，抗增殖和cerebroprotection的动作。所述AT 2 R也被确定为抗高血压和最近，抗癌药21的靶。确定各种组织内的血管紧张素Ⅱ受体的定位和密度以及他们是如何通过定量光密度的受体放射自显影将有助于揭开RAS在特定疾病中扮演的角色不同的治疗方法和疾病状态的影响。

受体放射自显影已经使用了30多年作为一种有效的方法，用于指示血管紧张素I的存在下1受体和大脑中的RAS的其它部件和大鼠，小鼠，豚鼠，在各种实验条件下22-34狗和人的其他组织。大脑内定位血管紧张素II受体的重要性是一个可以应用功能的神经解剖学至血管紧张素II的动作在大脑中， 例如 ，AT 1 R的下丘脑（PVN）的室旁核的存在表明昂的函数II在大脑刺激加压素，催产素或促肾上腺皮质激素释放激素（CRH）释放，或交感神经系统的活化。因此，阻断对AT 1 R药物可能会降低一些与在脑的RAS活性相关这些PVN介导的作用。正在进行的工作表明，AT 1 R拮抗剂的使用可以减少创伤后应激障碍（PTSD）的CRH诱导释放和缓解创伤后应激障碍的症状（伤害等 ，提交出版）。在PVN，穹窿器官（SFO），和杏仁核被称为调节体内平衡，食欲/口渴，睡眠，记忆，情绪反应，并且是本实证研究的目标区域。通过收集在显微镜玻片上脑的冠状切片，并治疗与特异性抑制剂的部分与血管紧张素II受体具有特异性放射性配体沿检查这些区域。在这项研究中，与建议的厂商沿所有材料和试剂中列出，碘-125，采用放射性标记的血管紧张素II受体拮抗剂，肌氨酸1，异亮氨酸8血管紧张素II（SI血管紧张素II），然后将其作为单声道125我纯化-SI血管紧张素II使用如前面所述的35 HPLC方法。使用这种高比活性的放射性配体的允许的放射性标记的部分暴露于X射线胶片后低，中和高受体密度的区域的可视化。通过与含有已知量的碘-125，具体量的脑糊标准校准膜血管紧张素II受体的区域结合的进行量化。在实验研究中，血管紧张素Ⅱ受体实验对象的大脑结合可以比对照受试者的大脑。这可以指示是否血管紧张素II的动作响应于遗传条件，表型异常，疾病状态或药物治疗被改变。然后，该知识可以应用到疗法来治疗与RAS的失调相关的疾病的发展。标识受体结合位点的替代技术，但具有降低的解剖分辨率，被结合使用从组织匀浆，这是与放射性配体的范围内的浓度，以评估作为解离常数放射性配体结合亲和力的培养来源的组织膜制剂测定（ 杀敌 ）和最大结合能力（B 最大值 ）的感兴趣的组织的。

这里所描述的协议可以被分解成5大共mponents：准备组织切片受体放射自显影;受体放射自显影;胶片曝光和发展;组织学;和密度图像分析。

Protocol

这项研究进行的所有动物的程序是由诺瓦东南大学的机构动物护理和使用委员会与指南实验动物的护理和使用一致认可， 第 8版（国家科学院出版社，华盛顿特区2011 ）。 1.准备组织切片受体放射自显影牺牲后，收获新鲜的脑组织，并尽快包装在铝箔并将其放置在-20℃的冰箱。要保持正确的形状，放置在大脑脑模具模拟头骨的内部，包裹在铝箔和发生在 – 20℃的冰箱中。 30分…

Representative Results

所述肾素-血管紧张素系统的代谢途径的概要示于图1和直接集中在血管紧张素II受体亚型（AT 1 R和AT 2 R）在图2中进行说明。 图3显示冠状脑转移节到显微镜载玻片，然后将其通过一个受体放射自显影方法，使用预定的125 I SIANG II浓度运行， 如图4所示。 图5示出了用于在受体放射自显影?…

Discussion

描述的协议标识“总”和“非特异性”在啮齿动物脑先前收获并保存在-80℃的冠状切片的相邻部分的放射性配体结合的可视化，并且可以容易地适用于几乎所有的组织中已解剖解决其中显示的受体或放射性配体结合位点的差的量子结构。在协议中描述的过程简单，分析是正确地解释结果的关键。测定20微米厚为最佳;如果部分过厚的非特异性结合会增加，从而难以解决的放射性配体的结合到它的目?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

This work was supported by NIH Grant HL-113905

Materials

500ml Plastic Beakers Fisher 02-591-30
24mm x 60mm Coverslips Fisher 22-050-25
Autoradiography Imaging Film 24x30cm Carestream-Biomax MR Film 891-2560
Bacitracin (from Bacillus licheniformis) Sigma B-0125
Cardboard Sheet 8×11 Crescent Illustration Board #201 201
Coplin Jars Fisher Scientific E94
Commercial hair dryers Conair Model SD6X
Disposable Culture Tubes Fisher 14-961-26
EDTA (Disodium salt, Dihydrate) USB Corporation 15-699
Ethanol Fisher 16-100-210 
Formulary Substitute for D-19 Developer Photographers Formulary, Inc.  01-0036
Glacial Acetic Acid Fisher A38 SI-212
Histoprep / OCT Fisher SH75-125D
Film Fixer Kodak 5160320
Photo flo Kodak 1464502
Losartan Fisher/Tocris 37-985-0
MCID™ Core 7.0 MCID N/A
NaCl Fisher S271
Peel-A-Way slide grips VWR 48440-003
Permount Fisher SP15-100
PD123319 Fisher 13-615-0
Premium Charged Slides , Fine Ground Edge Premiere Microscope Slides 9308W
125I Ligands Perkin Elmer NEX- 248
125SI-Ang II  George Washington University Radioiodinated by Dr. Speth
Slide Mailers Fisher Scientific HS15986
Sodium Dibasic Phosphate Anhydrous (Na2PO4) Fisher RDCS0750500
Sodium Acetate (Anhydrous) Fisher BP333-500
Thionin  Fisher T409-25
X-Ray Casette (10 x 12) Spectronics Corporation Four Square
Xylene Fisher  X3P-1GAL
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Tags

Receptor AutoradiographyAngiotensin II ReceptorsBrain SectionsQuantificationLocalizationNeurodegenerative DiseasesCardiovascular DiseasesHormone Receptor SystemsDrugs Of AbuseCryostat SectioningMicroscope SlidesRadio LabelingNon-specific TreatmentTotal Treatment
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Linares, A., Couling, L. E., Carrera, E. J., Speth, R. C. Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors. J. Vis. Exp. (112), e53866, doi:10.3791/53866 (2016).
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