作者感谢里斯凡Asselberghs和安凡Santvoort的出色技术援助。研究由内陆水运-VLAANDEREN（IWT SBO / 80020），该FWO VLAANDEREN（G.0768.10）资助，由EC-FP6项目"迪米"（LSHB-CT-2005-512146）中，FP7项目RTD MEFOPA（HEALTH -2009-241791），在FP7计划"INMiND"（健康F2-2011-278850），在鲁汶（IOF-KP / 07/001，OT / 08 / 052A，IMIR PF / 10/017），和MJFox基金会（目标验证2010）。 A.范德Perren和C Casteels是科学研究的弗拉芒基金的博士后。 K.范Laere是科学研究的弗拉芒基金的高级研究员临床。

所有的动物实验是由比利时鲁汶大学生物伦理委员会（比利时）按照1986年11月24日（86/609 / EEC）的欧洲共同体委员会指令进行批准。 1.重组腺相关病毒生产和纯化注意:rAAV载体生产和纯化由鲁汶病毒载体核（LVVC）如先前所述17进行。 <ol><li>简言之，转染汇合低（&lt;50）通道使用25KD线性聚乙烯亚胺150纳米的NaCl转染溶液和三种不同的质粒以1:1的比例粘附的HEK 293T细胞:1:1的DMEM培养基2％胎牛血清。后在37℃下在5％CO 2温育24小时，替换以新鲜的DMEM培养基2％胎牛血清的培养基。 注意:质粒包括用于AAV7血清型的构建体中，CMVIE的控制下，α-SYN编码人A53T突变的AAV转移质粒增强SYNAPSIN1子和pAdvDeltaF6腺病毒辅助质粒17。 </li><li>收获中5天瞬时转染后使用切向流过滤17集中。 </li><li>净化从浓缩介质使用碘克沙醇步骤梯度17 rAAV载体颗粒。 </li><li>使用实时PCR基因组副本（GC）测定的标准技术。在这个协议中，3.0 E11 GC的向量滴度/ ml的用于开发基于α-SYN大鼠模型PD 17。 </li></ol> 2.重组腺相关病毒α-SYN的立体定向注射载体在大鼠的SN（图2） <ol><li>众议院8周龄雌性Wistar大鼠可免费使用颗粒状的食物和自来水一个正常的12小时光/暗周期下重约200-250克</li><li>提交老鼠腹腔内（IP）含有氯胺酮（60毫克/千克），美托咪定（0.4毫克/公斤）混合物麻醉。一旦大鼠麻醉，并没有反应挤压不同爪子时，管理对大鼠的用微型发射机应答器植入背面为进一步识别微发射机应答皮下。检查，如果微发射机应答器被正确定位，并且可以通过读取装置读出。 </li><li>切头皮上的顶部的毛发。在头皮和耳都应用局部麻醉剂。使用无菌技术在层流下进行手术操作的其余部分。 </li><li>将使用两个耳朵酒吧，嘴和鼻子吧的立体定向头架的老鼠。覆盖纸毛毯的大鼠的体以避免在体温下降。应用眼部润滑剂，以防止眼睛变干。 </li><li>消毒在异丙醇70％与jodium 1％头皮和作一小切口在头皮的中线。轻轻刮掉头骨膜和用生理盐水冲洗。让头骨干几分钟。观察颅缝和两个参考点:前囟门和Lambda。 </LI&gt; <li>注入rAAV载体进入SN，定义坐标对前囟门（正位5.3毫米;内外侧斜:2.0毫米，背腹:从硬脑膜计算7.2毫米）。 注意:为每个感兴趣的区域的三维坐标可以使用大鼠大脑的立体图集进行计算，施加前囟如解剖参考点。 </li><li>填有10微升注射针筒（30计20毫米），腺相关病毒载体，并将其放置在与机动微量注射泵连接在立体定位仪。通过释放载体一滴控制音量和消除多元清洁剂pH值为9（ 如 RBS）。 </li><li>目视检查头直在头部框架固定和评估的左右轴。仔细直观定义前囟门和Lambda前后及中侧坐标和立体定位框架的背腹臂用30计20毫针刺测量自己的身高。 <ol><li>允许时在囟门和Lambda之间的高度0.3毫米差aximum。放置针回到前囟门和通过移动的前后和立体定位框架的中侧臂施加的前后和中侧坐标。 </li></ol></li><li>在注射位置，测量头骨的高度，并确保它不会从囟的高度相差大于0.3毫米。钻在头骨的孔的直径为约2毫米。测量硬脑膜的高度，这将作为应用背腹坐标参考。可替代地减去对颅骨（0.9毫米）的固定厚度。 </li><li>使用26号针头穿透硬脑膜，用无菌组织吸收血液。等到所有出血已在继续之前停止。 </li><li>慢慢插入10微升注射注射器预加载了载体溶液进入脑到预先确定的深度（背腹坐标）。等待1分钟，以代替针。注射3微升使用机动微量注射泵，吞吐量0.25微升（的rAAV2 / 7α-SYN或绿色荧光蛋白控制向量的3.0 E11基因组拷贝/ ml（中等矢量剂量）或1.0 E12 GC /毫升（高矢量剂量））向量解/分钟。 </li><li>注射后，保持针到位再过5分钟，慢慢地，再取出。使用拼接编织涂层聚酯3.0头皮，消毒用1％jodium在70％异丙醇和轻轻地从立体定向仪取出的动物。首先松开鼻子和嘴棒，那么这两个耳酒吧。 </li><li>扭转麻醉，用0.5毫克/千克腹膜内阿替美唑注入大鼠和放置老鼠在一个干净的笼子上的38℃的加热板，直到其被唤醒。覆盖纸毛毯大鼠防止体温下降。 </li><li>提供容易获得食物和水的第一个小时。监测的最初几天的老鼠。如果需要应用镇痛。 注意:没有必要去除从t线圈他的头骨。 1-2周后颅骨完全修复和拆线松动。 </li></ol> 3.的rAAV2评估/ 7α-SYN注射的大鼠使用非侵入性的PET显像，行为测试和免疫组化分析<ol><li>跟进黑质纹状体多巴胺神经变性的动力学非侵入过在动物个体时，量化多巴胺转运蛋白（DAT），使用小动物正电子发射断层扫描（PET）和DA转运如的示踪剂结合[18 F] -FECT 16 。 </li><li>审查多巴胺能神经变性的程度是否足以诱发在大鼠运动障碍，若使大鼠在气缸试验，评价自发前肢使用。 <ol><li>放置老鼠在期间沿后部后壁和到达垂直运动宽透明玻璃筒和录像带的行为20厘米分数由每个前爪做触点数量共计20联系人。对的评分标准的详细描述，参见Schallert 等人 18快速的受损前肢触点（ 例如左前爪），为总前肢接点的百分比（左加右前爪）的数目。 注意:在使用两个爪同样非损伤对照大鼠应在此测试得分50％左右。 </li></ol></li><li>进行免疫组织化学（IHC）分析来评估转基因表达和多巴胺能细胞损失的水平。 <ol><li>在不同的最终阶段，处死大鼠用戊巴比妥钠（60毫克/千克，IP）的过量，并用冷盐水随后在PBS 19 4％多聚甲醛执行心脏内灌注。固定的大脑在4℃过夜，并用振动切片机切成50μm厚冠状脑切片。 </li><li>使用抗α-SYN和酪氨酸羟化酶的抗体来分析α-SYN表达水平和乐自由浮动的部分进行IHC染色神经退行性疾病16德维尔。 </li></ol></li></ol>

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作者宣称，有没有利益实际或潜在的冲突。

有该协议中的几个关键步骤。载体滴度以及载体纯度直接影响该模型的表型结果。过度载体滴度或充分纯化的载体批次，可能会导致非特异性毒性。因此，使用高品质的矢量批次，适当的控制载体是必不可少的。此外，鼠的头在立体框架的精确定位和坐标的精确确定是在指定目标的黑质是必不可少的。在注射部位钻在头骨的孔后，它在针插入直入老鼠的大脑不接触任何边距是重要的。针应注射病?...

研究PD的病理生理学和开发新的治疗策略，对于动物模型可以非常类似于人PD的神经病理学，生理和运动症状的迫切需要。较高的预测值，我们就能更好地转化动物模型的新疗法给患者。 α-突触核蛋白的发现（α-SYN）在1997年第一PARK基因导致了第一个基因PD模型的发展。许多表达人野生型（WT）或突变（A30P，A53T）的转基因小鼠α-SYN已在过去十年中产生的。 α-SYN过表达的水平已被证明是在病理学的发展是至关重要的。另外，小鼠品系，内源性α-SYN的和是否全长或截短形式表示存在或不存在，播放（由洋红色和Chesselet 1详细审查）的作用。 WT和人类的几个临床突变体的过表达＃945; -SYN在转基因小鼠诱导α-SYN病理累积和神经元功能障碍2-6。然而，直到现在大多数转基因α-SYN的小鼠模型未能显示效果清晰多巴胺细胞损失和多巴胺相关的行为缺陷。 这种障碍是由黑质（SN）用病毒载体过表达α-SYN的直接针对克服。病毒载体是从病毒，可以很容易感染细胞，引入遗传物质引入其宿主基因组中，并迫使宿主细胞中复制的病毒基因组中，以产生新的病毒颗粒衍生的。病毒可以被工程化以保留其进入细胞并引入基因的能力的非复制的病毒载体。通过删除的病毒基因组的某些部分与由感兴趣的基因替换它们，载体的应用将导致在单轮感染而不会在宿主细胞中，通常被指定为"转导"复制。病毒载体Ç一个同时用于过表达和基因沉默。所表达的转基因可以是一个报告蛋白（ 例如绿色荧光蛋白或萤火虫萤光素酶）7，治疗性蛋白质的基因治疗应用8-10，或如我们将集中于在本文中，用于疾病建模11的疾病相关蛋白质-14。 病毒载体介导的基因递送提供了另一种方式来表达在CNS转基因与几个优点。利用当地的转基因传送，特定的大脑区域可以有针对性。此外，转基因的表达可以减少成年后的补偿机制发展过程中的风险过程中引起的。此外，可以在不同的物种和株被创建的模型。最后，不同的转基因可以很容易地进行组合。使用病毒载体，高转基因表达水平可以实现，因为该疾病发病和严重程度常常取决于曝光过度的水平，这可能是重要的ression。 根据不同的病毒数载体系统已经开发出来。载体系统的选择依赖于感兴趣的基因，基因表达，靶细胞和生物安全问题的需要的持续时间的大小。在大脑中，慢病毒（LV）的稳定的基因转移和重组腺相关病毒（腺相关病毒）载体现在被认为是选择的载体系统，因为它们导致在啮齿动物脑有效和长期的基因表达。对于SN的多巴胺能神经元（DN）特异性靶向，腺相关病毒载体已逐渐outcompeted由于其较高的效价和DN的转染效率的LV载体。 目前可用的最佳α-SYN基于啮齿动物模型已经从使用较新的AAV血清型的组合的方法显影（腺相关病毒1，5，6，7，8）和优化的载体构建体，滴度和纯度15,16。载体滴度以及载体纯度直接影响该模型的表型结果。过度载体滴度或充分纯化的载体批次，可能会导致非特异性毒性。因此，适当的控制向量是不可缺少的。在病毒载体生产，像素提升和纯化过程相当长的时间也投资已经证明必不可少获得可重复性和高品质的矢量批次。

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:391px;">Female 8 weeks old Wistar rats</td>
			<td style="width:227px;">Janvier</td>
			<td style="width:187px;">/</td>
			<td style="width:681px;">200-250 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine (Nimatek)</td>
			<td>Eurovet animal health</td>
			<td>804132</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Medetomidine (Dormitor)</td>
			<td>Orion-Pharma/ Janssen Animal Health</td>
			<td>1070-499</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Local anesthetic for scalp and ears: Xyloca&#239;ne 2% gel</td>
			<td>Astrazeneca</td>
			<td>0137-547</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Terramycine</td>
			<td>Pfizer</td>
			<td>0132-472</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bupr&#233;norphine (Vetergesic)</td>
			<td>Ecuphar</td>
			<td>2623-627</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Jodium 1% isopropanol</td>
			<td>VWR</td>
			<td>0484-0100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">stereotactic head frame</td>
			<td>Stoeling</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hamilton Syringe (30 gauge -20mm -pst 2)</td>
			<td>Hamilton/ Filter Service</td>
			<td>7803-07</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">atipamezole (Antisedan)</td>
			<td>Orion-Pharma/Elanco</td>
			<td>1300-185</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rAAV A53T &#945;-SYN vector</td>
			<td>LVVC, KU Leuven</td>
			<td>/</td>
			<td>https://gbiomed.kuleuven.be/english/research/50000715/laboratory-of-molecular-virology-and-gene-therapy/lvvc/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium pentobarbital (Nembutal)</td>
			<td>Ceva Sant&#233;</td>
			<td>0059-444</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microtome</td>
			<td>Microm</td>
			<td>HM650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rabbit polyclonal synuclein Ab</td>
			<td>Chemicon</td>
			<td>5038</td>
			<td>1:5000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rabbit polyclonal TH Ab</td>
			<td>Chemicon</td>
			<td>152</td>
			<td>1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lutetium oxyorthosilicate detector-based FOCUS 220 tomograph</td>
			<td>Siemens/ Concorde Microsystems</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">radioligand: 18F-FECT</td>
			<td>In house</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-dopa: Prolopa 125</td>
			<td>Roche</td>
			<td></td>
			<td>6mg/kg i.p.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, Glutamax</td>
			<td>Life Technologies</td>
			<td>N&#176; 31331-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Foetal bovine serum</td>
			<td>Life Technologies</td>
			<td>N&#176; 10270-106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">25 kD linear polyethylenimine (PEI)</td>
			<td>Polysciences</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OptiPrep Density Gradient Medium: Iodixanol</td>
			<td>Sigma</td>
			<td>D1556-250ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optimen</td>
			<td>Life Technologies</td>
			<td>N&#176; 51985-026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paxinos 1 watston steretactic atlas, fourth Edition</td>
			<td>Elsevier</td>
			<td>/</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an alpha-synuclein based rat model for parkinson's disease via stereotactic injection of a recombinant adeno-associated viral vector

为了研究帕金森氏病（PD）的分子途径，并制定新的治疗策略，科学调查依靠动物模型。的PD-相关基因的鉴定已经导致遗传的PD模型的发展。大多数转基因α-SYN的小鼠模型开发逐渐α-SYN病理，但未能显示效果清晰多巴胺细胞损失和多巴胺相关的行为缺陷。这一障碍，通过用病毒载体过量表达的PD-相关基因的黑质的直接靶向克服。使用病毒载体本地基因递送提供了一个有吸引力的方式来表达在中枢神经系统中的转基因。特定的大脑区域可以靶向（ 例如黑质），表达可在成人设置被诱导，并且可以达到高表达水平。此外，可以使用基于各种病毒不同载体系统。的协议概括了所有关键步骤来执行的病毒载体注射的大鼠的黑质开发用于帕金森氏病的病毒的基于矢量的α-突触核蛋白的动物模型。

实验的总体方案在图1中所描绘 A53Tα-SYN的重组腺相关病毒2月7日-介导的过度表达导致多巴胺相关运动障碍。 为了检验α-SYN过表达水平是否足以诱发在大鼠运动障碍，我们进行了大鼠的气缸试验，评价自发前肢使用（ 图3A）。 3周至注射后，一个显著运动功能障碍主要出现在接受剂量3.0...

Watch this Scientific Journal Video about Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector at JoVE.co

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

development-an-alpha-synuclein-based-rat-model-for-parkinson-s

Research

JoVE Journal

Medicine

13.2K 次观看.  KU Leuven. This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease. 

视频: Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

高效率转导肝癌细胞的重组腺相关病毒血清型3载体

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

科研

医学

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

1单侧损毁6  - 羟基多巴胺帕金森病小鼠模型的发展

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI成像帕金森病的神经肽质谱

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

多囊肾一种可能的斑马鱼型号:击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.

肾内注射<em&gt;大肠杆菌</em&gt;在肾性肾炎大鼠模型中

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

伤口的牙髓评价一个直接盖髓模型的开发和治疗小鼠中修复性牙本质形成

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

重组蛋白治疗慢性疼痛的研究进展

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

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 ...

Development of a Uterosacral Ligament Suspension Rat Model

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately 10%-20% of women undergo pelvic floor repair surgery to treat prolapse in the United States. PFD cases result in an overall $26.3 billion annual cost in the United States alone. This multifactorial condition has a negative impact on the quality of life and yet the treatment options have only dwindled in the recent past. One common surgical option is uterosacral ligament suspension (USLS), which is typically performed by affixing the vaginal vault to the uterosacral ligament in the pelvis. This repair has a lower incidence of complications compared to those with mesh augmentation, but is notable for a relatively high failure rate of up to 40%. Considering the lack of standard animal models to study pelvic floor dysfunction, there is an urgent clinical need for innovation in this field with a focus on developing cost-effective and accessible animal models. In this manuscript, we describe a rat model of USLS involving a complete hysterectomy followed by fixation of the remaining vaginal vault to the uterosacral ligament. The goal of this model is to mimic the procedure performed on women to be able to use the model to then investigate reparative strategies that improve the mechanical integrity of the ligament attachment. Importantly, we also describe the development of an in situ tensile testing procedure to characterize interface integrity at chosen time points following surgical intervention. Overall, this model will be a useful tool for future studies that investigate treatment options for POP repair via USLS.

Pelvic organ prolapse affects millions of women worldwide and yet some common surgical interventions have failure rates as high as 40%. The lack of standard animal models to investigate this condition impedes progress. We propose the following protocol as a model for uterosacral ligament suspension and in vivo tensile testing.

子宫骶韧带悬浮大鼠模型的开发

Pelvic organ prolapse (POP) is a common pelvic floor disorder (PFD) with the potential to significantly impact a woman's quality of life. Approximately ...

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

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