Name: neurodegeneration in an animal model of chronic amyloid-beta oligomer infusion is counteracted by antibody treatment infused with osmotic pumps
Uploaded: 2016-08-14T15:00:00
Duration: 10 min 19 s
Description: Watch this Scientific Journal Video about Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps at JoVE.com

We thank Caroline Bouchard from the animal facility for the rat work. A.S. holds a J.A. De S&#232;ve master fellowship, and B.P. a COPSE fellowship from the Universit&#233; de Montr&#233;al This work was funded by grants attributed to J.B. from FRQS-Pfizer and start-up funds from H&#244;pital du Sacr&#233;-Coeur de Montr&#233;al Research Center.

伦理声明：该项目的协议动物与加拿大议会关于动物饲养的准则获得来自总医院心圣心，蒙特利尔的动物护理委员会批准，符合要求。 1.导管立体定向手术前的准备工作<ol><li>切PE50导管（6厘米的长度），将用于连接与渗透泵套管（ 见图1A）。填PE50导管与人工脑脊液（ACSF）和密封该导管的两端。保持所述导管在4℃直至使用。 </li></ol> 2.植入插管通过立体定向<ol><li>执行在无菌条件下手术。高压灭菌所有的手术器械和材料。清洁立体定位仪，彻底的工作区域，并用70％乙醇溶液消毒。戴上口罩，头发帽子和无菌手套。</li><li>通过注入腹膜内（IP）氯胺酮和赛拉嗪的溶液（100毫克/千克和10毫克/公斤，分别）麻醉大鼠。用温和的捏脚趾检查后确认动作麻醉。 </li><li>手术前至少30分钟皮下（SC）给麻醉动物;注入的非类固醇抗炎药（1毫克/千克美洛昔康）。 </li><li>使用剪剃动物的头部和与葡萄糖酸洗必泰溶液2％和异丙醇2％三次皮肤消毒。对眼睛应用兽医眼药膏，以防止干燥时的麻醉下。 </li><li>将动物与耳酒吧立体框架上。固定在立体框架的保持臂一个插管。通过将鼻锥递送3％异氟烷在整个过程中注入（SC）局部麻醉剂（布比卡因（1.5毫克/千克）和利多卡因（1.5毫克/千克）在头部的顶部。麻醉是维护，整个手术视野应该是披了，但它并没有在这里完成，以更好地展示该技术。 </li><li>使在头部的顶部用手术刀3厘米的切口。安装4个夹子切口周围离开头骨清晰。采用圆尖剪刀，使一个袋（2×2 平方厘米）的动物的肩胛骨之间的皮肤下。 </li><li>刮用刀片颅骨骨膜。头骨上应用纱布如果出血。 </li><li>验证头骨是平的，立体框架上完全一致。为了确保头骨是平的，检查高度的前囟门和拉姆达坐标。取将被用作参考点前囟的坐标。 </li><li>从囟开始，计算将在海马双侧植入两个导向套管的坐标（正位： - 0.42厘米;中侧：±0.30厘米;背腹： -根据Paxinos和Watson大鼠脑图谱0.28厘米）29 。指示用标记在插管的位置。 </li><li>在两个套管的着床点来说钻颅骨孔（0.5毫米）。钻两个孔等大约5毫米的位置和下面的这些要点插入应用牙科粘固粉时将巩固套管螺丝。 </li><li>切预先充满脑脊液用手术刀在导管的一端，而在套管的角臂插入。固定用牙科水泥的第一套管。避免将牙齿周围的水泥的位置在第二套管。 </li><li>让2牙科水泥干 - 3分钟，然后取出从第一套管持有人的手臂，并固定第二套管在里面。重复步骤2.11和2.12。把虚拟套管两个引导套管。确保虚设和导向导管具有相同的长度，以防止组织浸润和阻止插管。 </li><li>插入在先前的动物的肩胛骨之间所作的口袋两个导管的自由端。取下夹子，并缝合了皮肤缝合线4-0。 注：顶部导向套管需要为即将到来的Aβo输液访问。 </li><li>取下立体框架的动物和放回到笼子里。在手术后的恢复，将笼在加热水毯，直到动物醒来。笼应在垫部分，所以如果需要的老鼠可以远离热移动。监控动物不断，直到它重新获得足够的意识，以保持胸骨斜卧。 </li><li>返回大鼠动物设施从手术中恢复的密切监控10天。 注意：不要返回已动过手术，以公司的其他动物，直到完全康复的动物。美洛昔康（1毫克/千克）在手术后治疗疼痛两天给予每天一次。 </li></ol> 3.渗透泵安装<ol><li>一天安装之前，填充渗透泵与6E10抗体（1毫克/毫升; 100微升）和对照IgG1根据在无菌条件下制造商的说明;抗体（100微升1毫克/毫升）。保持在无菌蒸馏水泵在37℃下过夜，以激活泵。 </li><li>麻醉用异氟烷3％大鼠安装渗透泵（7天0.5微升/小时）。肩胛骨之间剃并用葡萄糖酸洗必泰溶液2％和异丙醇2％皮肤消毒。使2厘米的切口与肩胛骨之间手术刀来定位连接到导向导管的PE50导管。 </li><li>切含有牙科用水泥用解剖刀的PE50导管的末端。在渗透泵连接到PE50导管，并在泵和导管连接处添加一些牙科水泥固定连接。 </li><li>用缝合线4-0紧紧缝合皮肤。摆在笼子里的供热水垫动物回来。从异氟烷麻醉（约5分钟）观察动物唤醒迅速。 注意：手术需要Appro公司ximately 5至10分钟。 </li></ol> 4.Aβo输液清醒和自由​​活动大鼠<ol><li>制备如先前报道的Aβo溶液（0.2微克/微升）。28,30允许Aβo动态和自发聚集在室温输液前1小时。 </li><li>对输液泵安装两个10微升注射器。用5微升无菌蒸馏水填充注射器。切两PE50导管至约60厘米的长度用手术刀。填用1ml注射器和21G针头用无菌蒸馏水两个导管。 </li><li>保持1ml的注射器在导管的一端，另一端连接到汉密尔顿注射器。卸下1ml的注射器和检查是否有导管内无气泡。 </li><li>在PE50导管的末尾插入套管内部。使用无菌蒸馏水，填写连接到PE50导管内插管起来TO1微升汉密尔顿注射器。使用p气泡ulling回活塞高达2微升。 </li><li>混合通过上下吹打使用粘附的最小的尖端的Aβo溶液。避免混合过程中形成气泡。拉回活塞高达3.5微升填充1.5微升Aβo解决内部套管。 </li><li>做之前和在两个导管进行气泡后标记线。这可作为持续输注的检查点。输液，把附近的输液泵笼并删除其封面。 </li><li>发生在一个依偎的老鼠，紧紧围绕刮其脖子上依偎的手臂固定大鼠头部。从两个导向套管除去虚设插管。插入先前在导向导管中制备的内部套管。验证它们是否完全插入并固定良好的引导套管的底部。 </li><li>从依偎释放大鼠和放回它在笼子里，限制竞争的压力。密切监测，以确保导管不扭曲toget她和输液做得好。 </li><li>打开输液泵。注入1微升Aβo溶液以0.1微升/分钟（10分钟）的速率。期间输液，检查从3.5微升注射器活塞移动至2.5微升，并且在这两个PE50导管气泡连续地移动。 </li><li>留在原地内部插管输液接连5分钟，以允许Aβo解决方案有效扩散。把老鼠回到依偎去除内部套管和封顶引导套管，防止注入液回流。使用虚拟插管，只是套管的角度臂前停止。返回动物在笼子里。 </li><li>在连续6天，每天天重复Aβo输液一次。断头安乐死的动物。 </li></ol>

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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=Intracerebroventricular+amyloid-beta+antibodies+reduce+cerebral+amyloid+angiopathy+and+associated+micro-hemorrhages+in+aged+Tg2576+mice.">Intracerebroventricular amyloid-beta antibodies reduce cerebral amyloid angiopathy and associated micro-hemorrhages in aged Tg2576 mice.</a> Proc. Natl. Acad. Sci. USA. 106, 4501-4506 (2009).</li><li>Papon, M. A., Whittington, R. A., El-Khoury, N. B., Planel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+and+anesthesia.">Alzheimer's disease and anesthesia.</a> Front. Neurosci. 4, 272 (2011).</li><li>Le Freche, 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=Tau+phosphorylation+and+sevoflurane+anesthesia:+an+association+to+postoperative+cognitive+impairment.">Tau phosphorylation and sevoflurane anesthesia: an association to postoperative cognitive impairment.</a> Anesthesiology. 116, 779-787 (2012).</li></ol>

Publication of this video-article is sponsored by ALZET Osmotic Pumps.

有一些需要特别注意这个协议中的关键步骤。当植入插管，避免将当其过液，以防止阻塞所述第二套管的孔牙科用粘固剂。它放置牙科用粘固剂在连接到泵的P50导管的自由端，以防止刺激和可能的炎症反应是很重要的。立体定位手术当天，用虚设套管是相同的长度引导套管，以避免阻塞插管。但是，在安装之后泵使用较短虚设插管该停止前套管的角臂，以允许从泵到海马溶液的适当输注。密切监测Aβo输液，并验证导管完成的气泡被注射期间连续移动。务必确保输液过程中注入套管完全插入引导套管。 如果问题是Aβ输液过程中遇到，确认内插管不会被阻断。如果是这种情况，通过内部插管冲洗无菌蒸馏水中。如果引导套管受阻，转动内部插管到导向套管。否则移动至内部插管上下。争中依偎可以是压力为大鼠，尤其是在第一天。为了减少动物的压力，我们建议操作和立体定位手术前habituate大鼠的依偎。 许多优点可以归结为这种新颖的和体内的方法灵活。事实上，Aβo的性质注入可输注之前被精确控制，而且不同类型的Aβ制剂（例如合成的VS脑衍生的Aβ溶液）可以被注入，以评估其体内神经毒性。这种模式也可以被用于研究机构由各种Aβ种类（ 例如 ，单体，低和高分子量醇igomers，原纤维）可在体内诱发神经毒性作用，以及怎么样免疫治疗可能会抵消在大脑中的有害影响。由于输注在清醒，自由移动的动物进行，有麻醉剂和信号通路的Aβo溶液之间没有混杂影响，如图以前的研究。32,33输液中自由移动的动物也与行为测试相容的任何时间之前和输注后。 Aβo和泵的安装输注可以在不同年龄段的动物才能确定Aβo老化过程中的作用和治疗。由于神经变性在输液部位的附近发生，突触和神经元损失可以以不同的和局部的大脑区域被诱导。 Aβo的抵押输液和控制（车辆或争夺Aβ）可以控制在同一动物中的​​任何变化。相反，Aβo或控制溶胶以及英可以双边在右侧被注入并在行为任务测试动物时左海马，例如。 Aβo和治疗输液可以同时进行或者交替泵可以Aβo输液后进行安装，以评估如果治疗Aβ沉积后生效。这里所描述的相同的协议，也可以做Aβo脑室注射时使用。 Aβo对细胞内信号通路的作用可以之前和相当短的时间框架内的神经元损失后进行评估。 Aβ输注的剂量和数量也可以调整，以获得更多或更少的严重的Aβ的致病性。 虽然非常通用的，这种技术具有一定的局限性。插管植入产生手术后组织和神经炎症中的最初几天的机械破坏。因此，重要的是开始Aβoinfu之前要等待手术后至少一周锡永，并添加适当的控制（车辆或不活动的扰频的Aβ注射）以考虑到这些事件。此外，只有Aβo小体积的可输注限制溶液的扩散。 该渗透泵代表了旨在防止Aβ诱导的神经退行性疾病药物的临床前验证了方便和独特的交付方法。由于免疫治疗与6E10抗体已被证明以前减少Aβ积聚在大脑中，31我们所用的6E10抗体作为证明的概念来验证我们新在体内的方法。在此模型中渗透泵的使用可能现在可用于开发新的疾病改善治疗，可以防止Aβo的临床前AD患者的沉积和神经毒性。

它最初提议在大脑中不溶性的Aβ种类的积累是中央对AD发病1,2然而，淀粉样斑块也在一些认知功能正常老年人检测3-7为了克服相关性较差斑块沉积和认知缺陷之间存在在AD，最新报道显示毒性可溶性Aβo在该疾病，其中关联与患者的临床状态好得多。8-14由于Aβ低聚过程的开始存在下非常动态的，有人建议，神经毒性是由不同的Aβo14-16诱导，而不是只有一个特定类型的低聚物。由于许多研究表明，Aβo可以启动之前，突触和神经元损失突触功能障碍，17-24当前理论表明，Aβ相关治疗可能是有效的早期AD，而不是在稍后阶段如在临床试验中迄今测试。 AD发病的一个标志特征是在疾病的后期观察到了大量的和广泛的细胞死亡，并在显著突触和神经元损失中局部脑区域观察时记忆障碍成为在临床水平检测到。从内嗅皮层（EC），以齿状回（DG）项目穿通在AD发病初期明显扰动。在25,26 AD的前驱状态时，轻度认知障碍（MCI）变得明显显著的细胞死亡在欧共体被检测以及在DG突触损失。25,26 虽然大量的证据已经精确定位可溶性Aβo在早期AD中的毒性作用，8-14Aβo诱导的神经变性的神经元培养物或器官脑切片培养观察已更具挑战性的动物模型重现。27大部分转基因的AD模型过表达β有淀粉样蛋白斑，tau蛋白磷酸化，突触缺失和记忆障碍。但27，这些模型已经成功要少得多，在AD患者的海马造型观察到的细胞死亡。为了克服这些技术问题，我们开发了一种基于可溶性Aβo脑内注射的典范。我们先前报道，反复可溶性Aβo海马输注引起逐步神经元丢失和tau蛋白磷酸化，与AD记忆力下降相关的两个病理特点。28在这里，我们展示了一种新的方法，使用套管测试AD的治疗专门设计用于Aβo同时输注与渗透泵Aβo抗体（6E10）的持续输注。 渗透泵提供直接在Aβo的输注部位的体内测试抗Aβo诱导的神经变性的任何抗体（或其他化合物）的效率的独特方式。因此，这些泵再版ESENT一个方便的工具来建立关于广告的潜在治疗药物的作用机制确凿的证据的概念。由于最近的报告指出，可溶于Aβo在AD的早期阶段的重要影响，对Aβo执导了许多治疗方法实际上是由学术和制药实验室测试。这种新颖的动物模型允许模拟在早期AD中观察到的突触和神经元损失，和渗透泵被用于连续地在Aβo输注部位特异性注入治疗剂。测试了在温和过去几年作为促使研究者发起预痴呆病人的试验，以中度AD患者治疗的重复性故障之前Aβo开始大量积累，并产生不可逆的脑损伤。在这种情况下，检测防止沉积和Aβo的因而神经毒性的新化合物可能是在临床前的患者的利益。

<table border="0" cellpadding="0" cellspacing="0" width="792">
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neurodegeneration in an animal model of chronic amyloid-beta oligomer infusion is counteracted by antibody treatment infused with osmotic pumps

下降海马依赖性显记忆（记忆事实和事件）是阿尔茨海默氏病（AD）的最早期临床症状之一。它是公认的突触损失和随后的神经退行性疾病是在公元记忆障碍最好的预测。最近的研究已经强调，开始在人脑积累大约10到15岁前的临床症状变得明显可溶性淀粉样蛋白β寡聚体（Aβo）的神经毒性作用。许多报告表明，可溶性Aβo在公元模型和人体与记忆障碍相关。在神经元和大脑切片文化观察Aβo引起的神经退行性疾病已更具挑战性在许多动物模型重现。这里显示的重复Aβo输注模式解决这个问题，并允许两个关键领域开发新的疾病治疗方法修改：生物识别标记来诊断早期AD，并确定分子机甲共享机制支撑Aβo引起的记忆障碍在AD的发病。由于可溶性Aβo聚集比较快成与患者的临床状态不佳不溶于关联Aβ纤维，可溶性Aβo新鲜烹制并在6天注射，每天一次，产生显着的细胞死亡的海马。我们采用套管专门设计用于Aβo同时输液和使用渗透泵海马Aβo抗体（6E10）的持续输注。 这种体内方法创新现在可以在临床前研究中使用来验证可能妨碍Aβo的预痴呆患者的沉积和神经毒性的新的AD治疗的效率。

Aβo的神经毒性作用被在植入插管Long-Evans大鼠研究 海马的DG。可溶性Aβo注射每天在连续六天。我们使用插管专门设计用于Aβo同时输注并在使用渗透泵（ 图1A）海马6E10或对照IgG1抗体的连续输注。用于免疫组织化学大鼠麻醉，用0.9％的NaCl经心脏灌注，并浸入4％多聚甲醛溶液的大脑为24小时48小时和15％的蔗糖溶液。自由浮动冠状切片（40微米）用0.3％的H 2 O 2处理30分钟，阻塞在1％山羊血清1小时，并在4℃下用抗泛Aβ抗体孵育过夜。切片施加抗体之前用70％甲酸处理3分钟。检测是使用复杂的ABC和0进行。05％3,3-二氨基联苯胺的解决方案。部分被安装在明胶包被的载玻片上，空气干燥2小时，脱水（30，70，95，和100％的醇），在甲苯5分钟温育，并盖上盖玻片。对甲酚紫染色，切片在0.5％甲酚紫溶液孵育10分钟，孵育在0.5％的乙酸溶液1分钟，脱色（70，95，和100％的醇），在甲苯中浸泡5分钟，并盖上玻璃盖玻片。 此处呈现的结果表明Aβo在DG中输液部位的附近与该累积相关联的沉积和细胞死亡。我们发现，水平Aβo基本上由6E10抗体处理（ 图1B）降低。观察Aβo的注入部位附近标记的神经变性，并用6E10抗体处理（ 图1B）衰减。这些结果与我们在DG观察Aβo积累一致小鼠经反复注射Aβo28清除淀粉样蛋白沉积的免疫所用这里显示的6E10抗体与在转基因AD小鼠模型所做的另一份报告线。31 <img alt="图1" src="/files/ftp_upload/54215/54215fig1.jpg" /> 图1. 双侧插管动物的照片在Aβo输液 Aβo的溶液。（0.2微克/微升1微升）用连接到内部套管PE50导管在清醒和自由活动的大鼠注射（每日一次6天）插入引导套管。与对照（CTL）的IgG1或6E10抗体治疗在使用位于皮下动物的肩胛骨之间渗透泵输注Aβo的部位直接注入。 <a href="https://www.jove.com/files/ftp_upload/54215/54215fig1large.jpg" target="_blank">请点击此处查看次的放大版本</a>是图。 <img alt="图2" src="/files/ftp_upload/54215/54215fig2.jpg" /> 。在连续6天后注射一天一次，和治疗与CTL（IgG1的）或其在6E10抗体; 可以通过Aβo沉积诱导 图2 的神经元损失是由6E10抗体治疗 A，Aβo（1微升0.2微克/微升） 减毒使用渗透泵输注。B，在DGAβo的代表性积累紧靠套管插入上的部分，和代表染色的部位与表示细胞损失甲酚紫。比例尺：50微米（N = 4） <a href="https://www.jove.com/files/ftp_upload/54215/54215fig2large.jpg" target="_blank">点击此处查看该图的放大版本。</a>

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Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

神经变性慢性淀粉样蛋白-β低聚物输注的动物模型是由抗体治疗灌注抵消与渗透泵

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

Research

JoVE Journal

Medicine

A Model of Chronic Nutrient Infusion in the Rat

在大鼠模型慢性营养输液

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

科研

医学

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

使用渗透皮下血管紧张素Ⅱ输液泵诱导主动脉瘤小鼠

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

免疫活性动物模型胶质母细胞瘤的生物发光成像

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

Continuous IV Infusion is the Choice Treatment Route for Arginine-vasopressin Receptor Blocker Conivaptan in Mice to Study Stroke-evoked Brain Edema

持续静脉注射是精氨酸加压素受体拮抗剂考尼伐坦的选择治疗途径小鼠来研究中风诱发脑水肿

Stroke is one of the major causes of morbidity and mortality in the world. Stroke is complicated by brain edema and other pathophysiological events. Among ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

定量3D<em&gt;在硅片</em&gt;建模阿尔茨海默病的啮齿动物模型脑淀粉样蛋白-β吞噬（q3DISM）

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

应用 Ameroid 的猪慢性心肌缺血模型

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection

万古霉素滴入硫酸钙和自体骨治疗兔骨感染的改进模型

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

慢性血栓栓塞性肺动脉高压大型动物模型中急性右心衰竭的诱导和表型分析

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

通过胆管 TNBS 输液治疗慢性胰腺炎的鼠标模型

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...