这项研究是由赠款R01NS062842从神经混乱国家学院和健康（VVD）的行程，国家机构和补助R21 NS064403从神经混乱国家学院和中风，国家通过ARRA卫生研究院（VVD）和R21支持生物医学成像和生物工程的EB006301研究所，美国国立卫生研究院（VVD）。

该方法适用于甲醛固定，石蜡包埋的组织切片。该技术是在这里的应用程序演示实验中风大鼠。 1，对酪胺信号放大（TSA）系统组件的准备虽然该协议为基础的强化中使用的标签的最后阶段，准备用于该反应可能需要超过1个小时。因此，这是方便的开始标签之前必须进行的全港性系统评估的试剂。 <ol><li>使荧光酪胺原液。使用与TSA标记试剂盒将各小瓶提供的荧光酪胺试剂。加入300μLDMSO的酪胺试剂瓶。荧光团的酪酰胺储备液是稳定至少3个月贮存于4℃。时</li><li>使含有TNT洗涤缓冲液：0.1M的Tris-HCl，pH值7.5; 0.15 M氯化钠; 0.05％Tween-20的。如果需要，0.3％的Triton X-100，可以被取代的0.05％吐温-20。交替结构延续PBS可以用作洗涤缓冲液。 </li><li>使TNB阻断缓冲液含有：0.1M的Tris-HCl，pH值7.5; 0.15 M氯化钠; 0.5％封闭试剂（与试剂盒提供）。以完全溶解的封闭试剂，慢慢地以小的增量添加到它的缓冲器，同时搅拌。热火这个解决方案逐渐到55℃，不断搅拌。这可能需要30-60分钟。该解决方案将出现乳白色。使用前将室温。分装和储存在-20℃下长期使用。 </li></ol> 2，准备的章节中使用幻灯片与安装5微米厚的切片切从福尔马林固定组织。 <ol><li>治疗部分用二甲苯15分钟。 </li><li>补充水分的部分如下：浸泡在96％乙醇2倍，每次5分钟;浸泡在80％乙醇进行5分钟;用清水洗净2倍，每次5分钟。 </li><li>使蛋白酶K工作溶液稀释的蛋白酶K原液至50μg/ ml的PBS中。 </li><li>添加蛋白酶ķ工作液部分。孵育15分钟，在23°C。溶液的体积和消化的时间可能需要根据部分和组织类型的大小进行调整。对于一个小的（小于5毫米的直径）第50微升蛋白酶溶液就足够了。对于一个平均大小（〜10毫米直径）部分，添加100微升的蛋白酶K溶液。对于非常大的部分中的溶液的体积可以按比例增加。在所有情况下，注意避免部分过量而导致完成信号的损失和破坏组织形态。这种情况经常发生，如果培养时间超过25分钟。 </li><li>在蒸馏水中2个，每次5分钟洗净切片。制备含有有源探头的标签组合，同时部分是水。 </li></ol> 3。VACC TOPO为基础的染色<ol><li>通过结合以下准备有源探头解决方案： 水 - 11.75微升 1米的Tris-HCl，pH值7.4 - 1.25μ升 100皮摩尔的12碱基悬自杀的寡核苷酸 - 1微升 100皮摩尔适配器寡核苷酸 - 1微升 50皮摩尔VACC TOPO - 10微升</li><li>通过移液轻轻混匀。在室温下孵育（23℃）搅拌15分钟，以允许探测器激活。 </li><li>采取切片洗罐和吸出剩余的水。然后应用在步骤3.1（元〜10×10mm的第25微升）编制的有源探头解决方案。 </li><li>盖玻片部分孵育15分钟，在23°C。在水中浸滑轻轻地去除盖玻片。在3倍的水，每次5分钟洗净。 </li></ol> 4，TSA信号增强<ol><li>涵盖组织切片与TNB封闭液提前准备，并培养玻片在湿盒中30分钟，在室温（23℃）。 </li><li>制备1:100稀释的链亲和素 - 辣根过氧化物酶结合物（SA-HRP）在TNB封闭缓冲液。吸TNB阻断缓冲液并应用SA-HRP解决方案。幻灯片孵育与SA-HRP 30分钟在室温下。使用适当的试剂量以弥补组织切片，每节一般在100微升。 </li><li>洗幻灯片3倍5每个在室温下最小的TNT洗涤缓冲液（或PBS）。 </li><li>准备1:50稀释的荧光酪胺股票在1x扩增稀释液的试剂盒。移液器的酪胺荧光工作液到每张幻灯片。使用足够的工作溶液中以完全覆盖组织部分，每部分通常为100微升。马上每个标签之前，请荧光酪胺的工作方案。该解决方案不能重复使用，所以标签后丢弃任何未使用的部分。 </li><li>孵育载玻片在室温下放置3到10分钟。监视显微镜下荧光增加直到结构的第一提示可以看出。赶紧进行清洗步骤。 </li><li>洗幻灯片3倍5每个在室温T MIN在TNT的洗涤缓冲液（或PBS）emperature搅拌。 </li><li>加用DAPI和盖玻片抗褪色的解决方案。在荧光显微镜下观察。可视化的DAPI和FITC荧光使用带通滤波器组： FITC激发D490/40，发射十分之五百二十; DAPI激发D360/40，发射二十○分之四百六，或类似的。 </li></ol>

<ol>
	<li>Jin, R., Yang, G., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mechanisms+in+ischemic+stroke:+role+of+inflammatory+cells.">Inflammatory mechanisms in ischemic stroke: role of inflammatory cells.</a> J Leukoc Biol. 87, 779-789 (2010).</li><li>Schilling, M., Besselmann, M., M&#252;ller, M., Strecker, J. K., Ringelstein, E. B., Kiefer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predominant+phagocytic+activity+of+resident+microglia+over+hematogenous+macrophages+following+transient+focal+cerebral+ischemia:+an+investigation+using+green+fluorescent+protein+transgenic+bone+marrow+chimeric+mice.">Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice.</a> Exp Neurol. 196 (2), 290-297 (2005).</li><li>Faustino, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cells+contribute+to+endogenous+brain+defenses+after+acute+neonatal+focal+stroke.">Microglial cells contribute to endogenous brain defenses after acute neonatal focal stroke.</a> J Neurosci. 31 (36), 12992-13001 (2011).</li><li>Thored, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+accumulation+of+microglia+with+proneurogenic+phenotype+concomitant+with+persistent+neurogenesis+in+adult+subventricular+zone+after+stroke.">Long term accumulation of microglia with proneurogenic phenotype concomitant with persistent neurogenesis in adult subventricular zone after stroke.</a> Glia. 57 (8), 835-849 (2009).</li><li>Sierra, A., Abiega, O., Shahraz, A., Neumann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Janus-faced+microglia:+beneficial+and+detrimental+consequences+of+microglial+phagocytosis.">Janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis.</a> Front Cell Neurosci. 7 (6), (2013).</li><li>Samejima, K., Earnshaw, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trashing+the+genome:+Role+of+nucleases+during+apoptosis.">Trashing the genome: Role of nucleases during apoptosis.</a> Nat.Rev. Mol. Cell Biol. 6 (9), 677-688 (2005).</li><li>Minchew, C. L., Didenko, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+probes+detecting+the+phagocytic+phase+of+apoptosis:+enzyme-substrate+complexes+of+topoisomerase+and+DNA.">Fluorescent probes detecting the phagocytic phase of apoptosis: enzyme-substrate complexes of topoisomerase and DNA.</a> Molecules. 16 (6), 4599-4614 (2011).</li><li>Shuman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+classes+of+DNA+end-joining+reactions+catalyzed+by+vaccinia+topoisomerase.">Two classes of DNA end-joining reactions catalyzed by vaccinia topoisomerase.</a> Sci. Rep. 267, 16755-16758 (1992).</li><li>Barcia, C., 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=ROCK/Cdc42-mediated+microglial+motility+and+gliapse+formation+lead+to+phagocytosis+of+degenerating+dopaminergic+neurons+in+vivo.">ROCK/Cdc42-mediated microglial motility and gliapse formation lead to phagocytosis of degenerating dopaminergic neurons in vivo.</a> Sci. Rep. . 2, 809 (2012).</li><li>Didenko, V. V., Ngo, H., Minchew, C. L., Boudreaux, D. J., Widmayer, M. A., Baskin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+irreparable+ischemic+damage+in+brain+by+selective+labeling+of+double-strand+blunt-ended+DNA+breaks.">Visualization of irreparable ischemic damage in brain by selective labeling of double-strand blunt-ended DNA breaks.</a> Mol. Med. 8, 818-823 (2002).</li></ol>

作者宣称，他们有没有竞争的财务权益。

在这段视频中，我们证明，在组织切片格式，如何标记吞噬细胞活跃其中透明细胞死亡局灶性脑缺血。所提出的技术是专门标签只有那些废物管理细胞已吞噬并积极消化细胞尸体的第一种方法。这使得它相对于对吞噬细胞，其不具有这种能力的其它标记的现有方法是有利的。 该方法使用吞噬活性的一般和选择性DNA为基础的标记 - 双链，钝核吞噬消化过程中废物管理细胞吞噬?...

缺血性中风引起的影响脑组织的深刻变化。它发起大量的细胞死亡，从而导致小胶质细胞来源的居民吞噬细胞迅速激活。它也由各种类型的血源性专业吞噬细胞包括中性粒细胞，巨噬细胞，树突状细胞和肥大细胞1,2衬托缺血性脑浸润。 目前仍有争论，这是否反应缺血性损伤起到积极的或消极的作用。虽然吞噬中风后，因为它清除死细胞和抑制炎症可以是有益的，它也产生有毒的活性氧自由基影响神经元的存活和加剧组织损伤1-5。 虽然几种类型的吞噬细胞浸润缺血性脑，不是所有的人都被吞噬细胞的尸体和再生过程1-3结算方式参与废物管理。此CReates选择性识别废物管理的细胞，进行细胞死亡在中风的吞噬细胞清除的要求。当成像这样的活性废物管理信元，它也是重要的回答他们是如何高效率地降低了吞噬细胞尸体的问题。在吞噬死亡细胞的DNA的有效和完全降解非常重要，因为它可以防止自身免疫和病理核材料6的释放。 在这里，我们提出了使用特定的DNA断裂为主动吞噬细胞的新标志物探针。这些签名里面休息职能废物管理细胞吞噬细胞核破裂过程中独家生产。因此，探针选择性标记，只有那些吞噬细胞吞噬，并积极消化细胞尸体。他们还允许观察吞噬后的强度和DNA分解完成。该探针是有用的在强度和effici的评价ENCY吞噬细胞清除的。 新探针依赖于非蛋白质为基础的标记，因此可以特别有利的行程，其中广泛的缺血性损伤可破坏细胞形态或耗尽基于蛋白质标志物，特别是内芯缺血区的研究。 该技术的原理是在图1，图中示出了由酶牛痘拓扑异构酶（VACC TOPO）连接发夹形的寡核苷酸探针 到5&#39;OH DNA末端的DNA消化7时溶酶体的DNase II产生。

<table border="0" cellpadding="0" cellspacing="0" width="696">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:312px;">Name of the reagent or material</td>
			<td style="width:99px;">Company</td>
			<td style="width:157px;">Catalogue number</td>
			<td style="width:129px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>JT Baker</td>
			<td>9490-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Roche</td>
			<td>3115879001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS Buffer</td>
			<td>Invitrogen</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vaccinia Topoisomerase I</td>
			<td colspan="2">Vivid Technologies</td>
			<td>5 pmol/&#956;L stock&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">12-Base Overhang Suicide Oligonucleotide</td>
			<td>IDT DNA</td>
			<td></td>
			<td>5&#8217;-AAG GGA CCT GCB GCA GGT CCC TTG ATA CGA TTC TA -3&#8217; ; B &#8211; Biotin-dT; 100 pmol/&#956;L stock in ddH2O&#160;&#160;</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Adapter Oligonucleotide</td>
			<td>IDT DNA</td>
			<td></td>
			<td>5&#8217;-TAG AAT CGT ATC-3&#8217;; 100 pmol/&#956;L stock in ddH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M Tris-HCl, PH 7.4</td>
			<td>Lonza</td>
			<td>51237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TSA, Fluorescein System&#160;</td>
			<td>PerkinElmer&#160;</td>
			<td>NEL701A001KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20&#160;</td>
			<td>Sigma</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Coverslips</td>
			<td></td>
			<td></td>
			<td style="width:129px;">22x22mm or 22x40mm glass or plastic coverslips. Plastic coverslips are preferable during the reaction, as they are easier to remove from the section.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">Rat Brain Sections</td>
			<td></td>
			<td></td>
			<td style="width:129px;">Rat brain taken 48 hours after reproduction of ischemic stroke as described in 11. 5-6&#956;m-thick sections cut from paraformaldehyde-fixed, paraffin-embedded tissue blocks. Use charged and precleaned slides.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Video camera and software</td>
			<td>Micromax</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software</td>
			<td>Metamorph</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing phagocytic clearance of cell death in experimental stroke by ligatable fluorescent probes

我们描述局灶性脑缺血的吞噬细胞清除的可视化一个新的组织化学方法。该方法允许消除死细胞的行程中任何细胞谱系的废物管理吞噬细胞的研究。虽然不同的起源，有能力吞噬了无数的细胞存在于脑缺血，其中只有一部分积极吞噬和消化细胞的尸体。选择性可视化，量化和这种积极的吞噬废物管理的分析是评估大脑反应缺血帮助。高效的细胞死亡的间隙是由缺血性脑损伤的恢复很重要，因为它开辟了道路，为后续的再生过程。未能清理尸体会导致因使用非降解的DNA和蛋白质毒性反应。所描述的过程使用的荧光探针由病毒拓扑异构酶选择性地连接到吞噬过程中所有的吞噬细胞产生的特征DNA断裂和细胞消化缺血不可逆的损坏。该方法用于脑反应缺血性损伤的调查的新工具。

该技术用于标记活性废物管理吞噬细胞的原理示于图1的示意图显示出，在三个步骤中检测所得。第一步骤包括探测激活（ 图1A）;在第二步骤中被激活的探测器被连接到在组织切片上（ 图1C）的特定DNA断裂;第三步骤包括荧光信号放大（ 图1D）。在一个更详细的解释，检测去如下： 1，探头激活 。之前，将连接反?...

Watch this Scientific Journal Video about Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes at JoVE.com

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

我们提出了选择性的一个新的荧光技术在主动吞噬细胞原位标记，它明确了在中风细胞的尸体。该方法对于评估脑反应缺血重要的，因为目前在缺血性脑吞噬细胞的只有一小部分参与细胞死亡的间隙。

评估细胞死亡的吞噬细胞清除由Ligatable荧光探针试验行程

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination ...

assessing-phagocytic-clearance-cell-death-experimental-stroke

Research

JoVE Journal

Medicine

8.1K 次观看.  Baylor College of Medicine. 我们提出了选择性的一个新的荧光技术在主动吞噬细胞原位标记，它明确了在中风细胞的尸体。该方法对于评估脑反应缺血重要的，因为目前在缺血性脑吞噬细胞的只有一小部分参与细胞死亡的间隙。 

视频: Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

建模在小鼠体内的行程 - 中东与灯丝模型大脑中动脉闭塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

科研

医学

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

共聚焦成像在肝癌细胞中观察到的异常有丝分裂细胞死亡相关

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

实验诱发青光眼通过磁性微球注射眼

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

胆管结扎小鼠：感应炎性肝损伤和纤维化的阻塞性胆汁淤积

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

玫瑰红Photothrombosis共聚焦光学成像<em&gt;在体内</em&gt;：单支脑卒中模型

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk factor for AD, stroke has also been implicated as a risk factor and an exacerbating factor. The co-morbidity of stroke and AD results in worsened stroke-related motor control and AD-related cognitive deficits when compared to each condition alone. To model the combined condition of stroke and AD, a novel transgenic rat model of AD, with a mutated form of amyloid precursor protein (a key protein involved in the development of AD) incorporated into its DNA, is given a small unilateral striatal stroke.
For a model with the combination of both stroke and AD, behavioral tests that assess stroke-related motor control, locomotion and AD-related cognitive function must be implemented. The cylinder task involves a cost-efficient, multipurpose apparatus that assesses spontaneous forelimb motor use. In this task, a rat is placed in a cylindrical apparatus, where the rat will spontaneously rear and contact the wall of the cylinder with its forelimbs. These contacts are considered forelimb motor use and quantified during video analysis after testing. Another cost-efficient motor task implemented is the beam-walk task, which assesses forelimb control, hindlimb control and locomotion. This task involves a rat walking across a wooden beam allowing for the assessment of limb motor control through analysis of forelimb slips, hindlimb slips and falls. Assessment of learning and memory is completed with Morris water maze for this behavioral paradigm. The protocol starts with spatial learning, whereby the rat locates a stationary hidden platform. After spatial learning, the platform is removed and both short-term and long-term spatial reference memory is assessed. All three of these tasks are sensitive to behavioral differences and completed within 28 days for this model, making this paradigm time-efficient and cost-efficient.

Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer&#039;s Disease with Stroke

To investigate the co-morbid Alzheimer&#39;s disease (AD) and stroke condition in a novel model, three behavior tasks are described that assess both motor control and cognitive behaviors. These tasks include the beam-walk task, cylinder task and Morris water maze.

汽车和海马依赖空间学习和记忆参考评估与中风阿尔茨海默氏病的转基因大鼠模型

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk ...