Fundação保护行使宪法权利支持的Pesquisa州里约热内卢（FAPERJ）和Conselho国立发展Científico发送Tecnológico圣保罗大学。我们非常感谢匿名审稿的意见，帮助我们改进的手稿和视频。

固体形式储存的所有化学物质，如6 - OHDA和阿朴吗啡，在无菌注射用水稀释和过滤层流罩内，以避免污染。此外，神经毒素和neuroactive物质储存，准备，处理和处置根据国际准则和当地生物安全委员会的规例。 1 - 麻醉和手术成人男性（25 - 30G）的瑞士小鼠premedicated与阿托品（0.2毫克/公斤，SC），以减少迷走神经张力，然后用氯胺酮（90-120毫克/公斤）和甲苯噻嗪（10毫克/公斤）腹腔注射麻醉。动物被放置在一个电子控制的加热垫（透视），以确保体温保持在37 ° C，作为镇静诱导一些分钟。整个过程中，通过测试撤退反射的情况下检查的麻醉水平。麻醉剂量应为每一株/殖民地ajusted。根据对动物和手术时间，额外剂量的氯胺酮可能是必要的。之后，生理盐水（氯化钠0.9％）是适用于两只眼睛，以防止晒出的角膜和测试眨眼反射的情况下在这第一时刻。在整个头部的毛皮剃光一个微调，以确保与未消毒的地区的最低接触。局部麻醉剂（Xylocaine）是适用于耳的酒吧，以防止不适。只有一次，撤回和眨眼反射已经消失，说明麻醉的水平是够深为手术（第3阶段），动物使用口片和耳酒吧专门为这个物种（透视）设计的框架上的立体定位。 positionated后，眼科软膏（马卡报）是适用于所有的手术，以保护整个动物的眼睛。只有在确保正确保护眼睛，程序继续重复三个70％的乙醇碘伏申请，与手术区域的皮肤消毒，用无菌棉拭子后，妥善安置耳棒确保通过测试头侧身运动（头不能移动，以确保酒吧是牢固地固定）。将核实后发现头骨的地标时，适当的头背腹倾向。一个矢状切口（1.5厘米），用无菌手术刀（＃15刀片）。骨膜在感兴趣的领域是轻轻地用一个新的刀片刮掉，骨擦干净，用无菌的目光和棉签，发现头骨的地标：前囟点的矢状面和冠状缝合线的交点定义，而波长矢状缝，通过左，右部分的人字缝的最合适的线路的交汇点。首先是一个垂直排列的参考针的尖端归零前囟门，通过手术显微镜下检查，然后移动触摸的lambda点。如果头是正确定位，λ应背腹为前囟在同一水平，并应沿rostro尾椎轴由4.2毫米遥远的两个地标。如果没有，耳朵的酒吧和消防水枪应放松和头部周围的双耳轴微妙的倾斜，以达到所需的位置，它定义了“扁平的头骨”stereotaxical措施的参考。参考针的尖端，然后在所需的坐标定位：前后（AP 0.5毫米）和横向（L -2.0毫米，左）。的位置上标注的头骨和一个小的（直径1.2毫米）的孔打开，用消毒的钻头，采用间歇性的行动，以防止传热面积。牙齿耳挖小心取出碎骨，并用无菌温暖的0.9％氯化钠冲洗。消毒5μL汉密尔顿注射器（26S压力表; 0.47毫米外径）装有6 - OHDA溶液（或控制动物的车辆），在立体定位仪和垂直对齐。针尖（类型2或12 °斜角），然后打开孔插入，直到触摸软脑膜表面，测定背腹坐标的参考点。一旦确定的坐标，针是慢慢降低，达到纹状体的坐标（美联社：0.5，L：-2.0和DV：-3.0毫米，软脑膜表面以下7 ）。 6 - OHDA溶液（10微克6 - OHDA以0.02％的抗坏血酸的0.9％氯化钠），这是准备和过滤层流罩内，并必须避光铝箔整个过程中，再注入一个0.1μL/min的流量。对于控制动物，车辆等体积（0.02％抗坏血酸和0.9％氯化钠无菌水）注入。一旦被注入，整个2.0μL，注射器保持在5分钟的地方之前，非常缓慢地从大脑缩回，在不超过5分钟。头骨是清理和关闭切口用6-0号尼龙缝合（Shalon）。采取一个额外护理防止感染，一个局部抗生素软膏，如新霉素，，可用于缝合皮肤。动物的框架，从0.5毫升生理盐水（sc.），以防止脱水，并保持温暖和密切观察下，直到完全恢复。然后，他们采用与食物和水随意。止痛药添加到手术后的头两个天（平均每天5.5毫升的水耗，饮水瓶和推荐剂量的布洛芬0.03毫克/克体重/日;添加0,82毫升20毫克/毫升布洛芬雅培暂停饮水100毫升）。鼠标通常程序及时恢复，徘徊和水食物颗粒很容易达到。在任何明显不适，或任何感染的迹象的情况下，动物，应当安乐死根据当地的指引。 2 - 行为学测试虽然运动功能障碍，可观察到一个星期后，可靠的量化与旋转试验前的1个月后病灶的间隔是允许的。动物收到的DA受体激动剂阿扑吗啡（0.5毫克/公斤）皮下注射，是摆在一个直径30厘米的不透明缸，放在45厘米以下的录音摄像头。重要的是要在每次测试之前删除任何残留物，并从以前测试过的动物的气味。经过5分钟的习惯，动作都录得超过5分钟的时间内。禁忌和同侧全身旋转测量和比较，与对照组（车辆注射）。如果程序正确执行，控制动物不会表现出任何旋转，而6 - OHDA损毁的动物将开始转向对侧，因为主要的激动剂激活过敏的神经支配纹状体损毁侧。得分超过700转（显示每分钟至少7全身对侧旋转）的动物被视为成功损毁。动物可以安全地返回测试后，每周可重复他们的住房30分钟。在我们的手中，不到5％的损毁小鼠显示不理想的旋转速率。

<ol>
	<li>Ungerstedt, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=6-hydroxy-dopamine+induced+degeneration+of+central+monoamine+neurons.">6-hydroxy-dopamine induced degeneration of central monoamine neurons.</a> Eur. J. Pharmacol. 5, 107-110 (1968).</li><li>Akerud, P., Canals, J. M., Snyder, E. Y., Arenas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotection+through+delivery+of+glial+cell+line-derived+neurotrophic+factor+by+neural+stem+cells+in+a+mouse+model+of+Parkinson's+Disease.">Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson's Disease.</a> J. Neurosci. 21, 8108-8118 (2001).</li><li>Ghosh, A., Roy, A., Liu, X., Kordower, J. H., Mufson, E. J., Hartley, D. M., Ghosh, S., Mosley, R. L., Gendelman, H. E., Pahan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+inhibition+of+NF-kappaB+activation+prevents+dopaminergic+neuronal+loss+in+a+mouse+model+of+Parkinson's+Disease.">Selective inhibition of NF-kappaB activation prevents dopaminergic neuronal loss in a mouse model of Parkinson's Disease.</a> Proc. Natl. Acad. Sci. USA. 104, 18754-18759 (2007).</li><li>Alvarez-Fischer, D., Henze, C., Strenzke, C., Westrich, J., Ferger, B., H&#246;glinger, G. U., Oertel, W. H., Hartmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+striatal+6-OHDA+model+of+Parkinson's+disease+in+wild+type+and+a-synuclein-deleted+mice.">Characterization of the striatal 6-OHDA model of Parkinson's disease in wild type and a-synuclein-deleted mice.</a> Exp. Neurol. 210, 182-193 (2008).</li><li>Ungerstedt, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=6-Hydroxydopamine-induced+degeneration+of+the+nigrostriatal+dopamine+pathway:+The+turning+syndrome.">6-Hydroxydopamine-induced degeneration of the nigrostriatal dopamine pathway: The turning syndrome.</a> Pharmacology and Therapeutics Part B: General and Systematic Pharmacology. 2, 37-40 (1976).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidelines for the Care and Use of Mammals in Neuroscience and Behaviour Research. , 209 (2003).</li><li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li><li>Hudson, J. L., Horne, C. G. v. a. n., Str&#246;mberg, I., Brock, S., Clayton, J., Masserano, J., Hoffer, B. J., Gerhardt, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+apomorphine-+and+amphetamine-induced+turning+with+nigrostriatal+dopamine+content+in+unilateral+6-hydroxydopamine+lesioned+rats.+Brain+Res.">Correlation of apomorphine- and amphetamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesioned rats. Brain Res.</a> , 626-6167 (1993).</li><li>Truong, L., Allbutt, H., Kassiou, M., Henderson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+a+preclinical+model+of+Parkinson's+disease:+A+study+of+behaviour+in+rats+with+graded+6-OHDA+lesions.">Developing a preclinical model of Parkinson's disease: A study of behaviour in rats with graded 6-OHDA lesions.</a> Behav. Brain Res. 169, 1-9 (2006).</li><li>Harvey, B. K., Wang, Y., Hoffer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+rodent+models+of+Parkinson's+disease.">Transgenic rodent models of Parkinson's disease.</a> Acta Neurochir. (Wien.). 101, 89-92 (2008).</li><li>Isacson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ole+Isacson:+Development+of+New+Therapies+for+Parkinson's+Disease.">Ole Isacson: Development of New Therapies for Parkinson's Disease.</a> JoVE. 3, (2007).</li></ol>

实验是在按照哺乳动物的关怀和使用的国际准则，在神经科学与行为研究<sup&gt; 6</sup&gt;之后，机构的动物护理和使用委员会的批准（Comissão DE Avaliação USO DE Animais EM Pesquisa：UFRJ / CAUAP协议＃DAHEICB 027）。采取适当措施，以尽量减少实验动物的痛苦和不适。

尼格罗 - 纹状体通路广泛的单方面病变可靠，可以实现在小鼠纹状体由一个6 - OHDA立体定向注入。病变范围可核实验尸报告由酪氨酸羟化酶免疫组织化学（催化DA的合成限速阶段，即L -酪氨酸，以二羟基苯转换），视频所示。 在体内，由阿朴吗啡诱导的对侧旋转是一个最大纹状体病变优于安非他明诱导同侧旋转8的预测。因此，可以监控功能的病变后果，在几个月内由?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ketamine hydrochloride</td>
<td>&nbsp;</td>
<td>K&ouml;nig</td>
<td>&nbsp;</td>
<td>90-120 mg/Kg</td>
</tr>
<tr>
<td>Xylazine hydrochloride</td>
<td>&nbsp;</td>
<td>K&ouml;nig</td>
<td>&nbsp;</td>
<td>10 mg/Kg</td>
</tr>
<tr>
<td>Atropine Sulfate</td>
<td>&nbsp;</td>
<td>Isofarma</td>
<td>&nbsp;</td>
<td>Isofarma</td>
</tr>
<tr>
<td>6 &#x2013; Hydroxydopamine Hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>5 &mu;g/&mu;L</td>
</tr>
<tr>
<td>L - Ascorbic Acid</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>&nbsp;</td>
<td>0.2%</td>
</tr>
<tr>
<td>Xylocaine (Lidocaine Chloridrate)</td>
<td>&nbsp;</td>
<td>ASTRA Chemistry</td>
<td>&nbsp;</td>
<td>5% 50 mg</td>
</tr>
<tr>
<td>Nebacetin (Neomycin Sulfate + Bacitracin)</td>
<td>&nbsp;</td>
<td>Altana Pharma</td>
<td>&nbsp;</td>
<td>5 mg/g + 250 Ul/g</td>
</tr>
<tr>
<td>R-(-)-Apomorphine hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>0.5 mg/Kg</td>
</tr>
<tr>
<td>GenTeal lOphthalmic ointment Hypromellose</td>
<td>&nbsp;</td>
<td>NOVARTIS</td>
<td>&nbsp;</td>
<td> 0.33%</td>
</tr>
<tr>
<td>Ibuprofen</td>
<td>&nbsp;</td>
<td>Abbott</td>
<td>&nbsp;</td>
<td>4 mg/ 100 ml drinking water</td>
</tr>
<tr>
<td>Povidine</td>
<td>&nbsp;</td>
<td>JohnsonDiversey</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>R-(-)-Apomorphine hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>0.5 mg/Kg</td>
</tr>		</tbody>
		</table>
		</div>

murine model for parkinson's disease: from 6-oh dopamine lesion to behavioral test

帕金森氏病（PD）影响全球至少有6.5亿人，不论性别，社会，种族，经济，或地理的界限。主要症状，如震颤，僵硬和bradikinesia，开发时在黑质多巴胺能神经细胞的3 / 4丢失，无法提供流畅，纹状体电机电路的协调监管。抑郁症和幻觉是常见的，和老年痴呆症在20％的患者最终发生。在这个时候，没有任何推迟或停止治疗PD的进展。相反，目前可用的目标，对减轻这些症状的药物更多。新的手术策略可能可逆开关电路通过电刺激脑深部结构的功能受损，但脑深部电刺激虽然是一个重大的进步，它不适合所有患者。因此，它仍然要测试新的细胞治疗方法在临床前模型。 选择性多巴胺能通路的神经毒性中断可以被复制，而消耗的药物和氧化损伤的化学物质注射6 - 羟多巴胺（6 - OHDA）或MPTP（1 - 甲基-4 - 苯基- 1 -1,2,3,6 - tertahydropyridine）也重现在啮齿类动物中的特定功能的PD。 MPTP的不同，6 - OHDA病变导致大规模的不可逆的神经元丢失，可单向或双侧。 6 - OHDA病变模型是可靠的，强大的运动障碍，并在rats1经过40多年的研究中使用最广泛的。由于移植的细胞和主机之间的相互作用现在可以在小鼠体内，而不是在大鼠的研究更彻底，该模型已被调换到2,3小鼠，它已被最近的特点4。 在这段视频中，我们将演示如何病灶慢慢通过stereotaxically插入微型注射器针头提供2.0μL6 - OHDA左尼格罗 - 纹状体通路麻醉小鼠。多巴胺输入的损失发生在几天之内，可以通过手术后的数周和数个月按等级动物多巴胺剂5诱导的旋转监控功能障碍。在这里，我们显示全身对侧旋转，10分钟后发生的一个单一的皮下注射阿朴吗啡管理，测量病灶后1个月。下面讨论的成果和缺点。

Watch this Scientific Journal Video about Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test at JoVE.com

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

帕金森病是造成损失，可实验6 - OH -多巴胺诱导的纹状体多巴胺能神经支配的。我们描述了如何执行的立体病变和监测阿朴吗啡诱导的小鼠旋转行为。这种模式是有用的，可靠的测试对帕金森病的新疗法。

帕金森氏病的小鼠模型：从6 - OH多巴胺病变行为测试

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

murine-model-for-parkinson-s-disease-from-6-oh-dopamine-lesion-to

Research

JoVE Journal

Biology

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

31.4K 次观看.  Universidade Federal do Rio de Janeiro, Brasil. 帕金森病是造成损失，可实验6 - OH -多巴胺诱导的纹状体多巴胺能神经支配的。我们描述了如何执行的立体病变和监测阿朴吗啡诱导的小鼠旋转行为。这种模式是有用的，可靠的测试对帕金森病的新疗法。

视频: Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson: Development of New Therapies for Parkinson&#039;s Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

OLE Isacson：帕金森氏病的新疗法的发展

科研

生物学

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

非洲爪蟾作为一种模型来确定翻译减值

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

子宫内膜异位症异种小鼠病变大小的无创监测

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). The most common pattern observed in the clinic is the peak-dose dyskinesia which consists of clinical manifestations of choreiform, dystonic, and ballistic movements. The 6-hydroxydopamine (6-OHDA) rat model of PD mimics several characteristics of LIDs. After repeated L-DOPA administration, 6-OHDA-lesioned rats exhibit dyskinetic-like movements (e.g., abnormal involuntary movements, AIMs). This protocol demonstrates how to induce and analyze AIMs in 6-OHDA-lesioned rats with 90%-95% dopaminergic depletion in the nigrostriatal pathway. Repeated administration (3 weeks) of L-DOPA (5 mg/kg, combined with 12.5 mg/kg of benserazide) can induce the development of AIMs. The time course analysis reveals a significant increase in AIMs at 30-90 min (peak-dose dyskinesia). Rodent models of LIDs are an important preclinical tool to identify effective antidyskinetic interventions.

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson&#039;s Disease

Rodent models of L-DOPA-induced dyskinesias are invaluable tools to identify therapeutic interventions to attenuate the development or alleviate the manifestations that emerge due to the repeated administration of L-DOPA. This protocol demonstrates how to induce and analyze dyskinetic-like movements in the unilaterally 6-OHDA-lesioned rat model of Parkinson's disease.

评价L-DOPA诱导的运动障碍在帕金森病单侧6-OHDA病变大鼠模型中

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). ...

小鼠肠隐窝和单干细胞类器官的 3D 培养

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

作者聚焦：来自小鼠肠隐窝的类器官的 3D 培养和用于类器官研究的单个干细胞  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

小鼠肠隐窝和单个干细胞的类器官的3D培养用于类器官研究

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...