我感谢 Dr. 艾伦· o. Perantoni (CDBL/国立卫生研究院/NIH) 支持这份手稿的提交。我还要感谢 Dr. 迈克尔霍尔 (CDBL/国立卫生研究院/nih) 和尼玛拉夏尔马 (CDBL/国立卫生研究院/nih) 编辑这篇手稿。我很感谢赖昌星 (SAIC) 的出色的鼠标饲养。这项工作得到了国立卫生研究院、国家癌症研究所和癌症研究中心的支持。

鼠标 ( Wnt5a flox / flox 小鼠 (Wnt5a tm1.1Tpy ) 和 Dll1Cre 线, UVJO 小鼠模型) 7 是根据 NIH 关于实验室动物护理和使用的指导方针进行管理的。并根据由动物保护和使用委员会批准的议定书进行研究. 
 1. 亚甲基蓝染料溶液的制备 <ol> <li> 测量 0.1 g 亚甲蓝粉. </li>
	<li> 在10毫升生理盐水或1x 磷酸盐缓冲盐水 (PBS) 中完全由涡流溶解亚甲基蓝. </li>
	<li> 用注射器过滤器 (膜孔径0.45 和 #181; m) 过滤10毫克/毫升亚甲基蓝溶液, 以消除注射过程中的堵塞. </li>
	<li> 组装一个无菌头皮静脉集 (27GX3/4 和 #8243;) 与3毫升一次性注射器和填充注射器3毫升过滤亚甲蓝溶液. 
	注意: 为了防止静水压和随后的染料流动, 将针尖放在装有亚甲基蓝溶液的注射器上. </li>
</ol> 2。产前胚胎解剖 <ol> <li> 用70% 乙醇清洗解剖剪刀和镊子. </li>
	<li> 在 E18.5 或 E19.5 中收集围产期胚胎, 先使用 CO 2 吸入, 然后根据 NIH 指南执行宫颈脱位. 
	注意: 怀孕的雌性老鼠通常从 E18.5 开始分娩, 但是, 较大的肾脏更容易操作。因此, 在 E19.5 接近出生的肾脏更容易进行这一分析。然而, 有双侧尿路阻塞的幼崽在出生后死亡。根据实验小鼠的特点, 应该经验地确定适当的收集日/时间. </li>
	<li> 在腹腹表面喷洒70% 乙醇, 然后用解剖剪刀和镊子打开腹腔腹. </li>
	<li> 抬起整个子宫, 用解剖剪刀在子宫角上切开, 将它与身体分开. </li>
	<li> 在培养皿中用 1x PBS 冲洗整个子宫. </li>
	<li> 用解剖剪刀切开子宫分段, 用解剖钳切除胎盘蜕膜, 将胚胎暴露在卵黄囊中. </li>
	<li> 先取下蛋黄囊, 然后用解剖钳取出羊膜, 释放围产期胚胎. </li>
	<li> 用解剖剪刀斩首胚胎。用无菌纱布擦拭多余的血液。在解剖显微镜上, 用数码相机进行成像, 将胚胎从腹面上固定下来。如果需要, 收集它的基因分型的尾巴. 
	注: 一次进行一次胚胎注射. </li>
	<li> 通过撕裂皮肤小心地打开一个胚胎的腹腔。然后, 通过切割或拔出它们来暴露肾脏、输尿管和位于背的膀胱 ( 图 1A ), 小心地清除多余的器官和组织, 如肝脏、胃和肠道。如有必要, 从解剖胚中吸收多余的血液, 用无菌纱布垫. 
	注: 过量的血液会影响到对染料注射的肾盂识别. </li>
</ol> 3。注射亚甲基蓝染料到肾盂和监测染料流 <ol> <li> 通过将亚甲基蓝溶液从针尖上排出, 通过静水压来除去针头和油管中的气泡。提起含有染料溶液的注射器, 在针尖的水平以上开始流动, 然后放下注射器停止流动. </li>
	<li> 将针插入靠近输尿管近端的肾盂, 注意不要在放置后再将其干扰。在肾脏中进行染料注射以确定其尿路梗阻. 
	注: 将染料注射到任何异常的肾脏 7 首先遵循同样的方法来确定其尿路梗阻. </li>
	<li> 抬起注射器约20厘米, 以提供静水压, 并让15和 #181; l 60 和 #181; 染料溶液流动的 l。流速将是大约3和 #181; 如果注射器是上升到这个高度在胚胎之上. </li>
	<li> 监测染料的蓝色颜色首先在肾盂中开始, 然后在输尿管的长度, 最后在膀胱腔. 
	注: 如果将输尿管正确插入膀胱内, 则需要大约5秒才能看到在膀胱腔中出现微弱的染料颜色。如果没有染料的颜色出现在膀胱腔中, 允许染料在被堵塞的地方堆积十五年代. </li>
	<li> 将注射器放下以停止染料流并从肾脏中取出针头. </li>
	<li> 使用与显微镜连接的照相机和成像程序, 将肾脏、输尿管和膀胱的图像与染料溶液一起跟踪. </li>
	<li> 将肾脏、us 和染料溶液的最终位置记录在实验室笔记本中. </li>
	<li> 执行3.1 节中描述的对侧肾脏的注射) 到 3.5), 并允许染料流在二十年代总. 
	注: 膀胱腔内将充满染料溶液, 以强烈的蓝色表示, 如果输尿管被正确插入膀胱腔。经尿路结肠梗阻评估, 输尿管和膀胱可固定切片. </li>
</ol>

<ol>
	<li>Rasouly, H. M., Lu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+urinary+tract+development+and+disease.">Lower urinary tract development and disease.</a> Wiley Interdiscip Rev Syst Biol Med. 5, 307-342 (2013).</li><li>Kispert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-Box+Genes+in+the+Kidney+and+Urinary+Tract.">T-Box Genes in the Kidney and Urinary Tract.</a> Curr Top Dev Biol. 122, 245-278 (2017).</li><li>Bohnenpoll, T., Kispert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ureter+growth+and+differentiation.">Ureter growth and differentiation.</a> Semin Cell Dev Biol. 36, 21-30 (2014).</li><li>Nicolaou, N., Renkema, K. Y., Bongers, E. M., Giles, R. H., Knoers, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic,+environmental,+and+epigenetic+factors+involved+in+CAKUT.">Genetic, environmental, and epigenetic factors involved in CAKUT.</a> Nat Rev Nephrol. 11, 720-731 (2015).</li><li>Tripathi, P., Wang, Y., Casey, A. M., Chen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+canonical+Smad+signaling+in+ureteral+and+bladder+mesenchyme+causes+ureteropelvic+junction+obstruction.">Absence of canonical Smad signaling in ureteral and bladder mesenchyme causes ureteropelvic junction obstruction.</a> J Am Soc Nephrol. 23, 618-628 (2012).</li><li>Aoki, Y., 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=Id2+haploinsufficiency+in+mice+leads+to+congenital+hydronephrosis+resembling+that+in+humans.">Id2 haploinsufficiency in mice leads to congenital hydronephrosis resembling that in humans.</a> Genes Cells. 9, 1287-1296 (2004).</li><li>Yun, K., Perantoni, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydronephrosis+in+the+Wnt5a-ablated+kidney+is+caused+by+an+abnormal+ureter-bladder+connection.">Hydronephrosis in the Wnt5a-ablated kidney is caused by an abnormal ureter-bladder connection.</a> Differentiation. 94, 1-7 (2017).</li><li>Uetani, N., Bouchard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plumbing+in+the+embryo:+developmental+defects+of+the+urinary+tracts.">Plumbing in the embryo: developmental defects of the urinary tracts.</a> Clin Genet. 75, 307-317 (2009).</li><li>Murawski, I. J., Watt, C. L., Gupta, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+urinary+tract+defects+in+mice:+methods+to+detect+the+presence+of+vesicoureteric+reflux+and+urinary+tract+obstruction.">Assessing urinary tract defects in mice: methods to detect the presence of vesicoureteric reflux and urinary tract obstruction.</a> Methods Mol Biol. 886, 351-362 (2012).</li><li>Weiss, A. 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=Nephric+duct+insertion+requires+EphA4/EphA7+signaling+from+the+pericloacal+mesenchyme.">Nephric duct insertion requires EphA4/EphA7 signaling from the pericloacal mesenchyme.</a> Development. 141, 3420-3430 (2014).</li></ol>

作者没有什么要透露的。

老鼠肾脏是功能开始在 E16.5 和染料射入测试从理论上可能从这个时间点。然而, 肾脏太小, 不能被注射的染料溶液和表型, 如肾积水和 us 没有清楚地观察到, 因为这些表型是由于尿液转运异常而产生的二次作用。这些表型, 从障碍, 如 UPJO 或 UVJO, 是明显的 E18.5 肿胀肾脏和输尿管。E19.5 或新生幼崽的胚胎肾脏比 E18.5 或更年轻的胚胎更容易操作。然而, 有双侧尿路阻塞的幼崽在出生后死亡。因此, 根据?...

泌尿系统包括一对肾脏和输尿管和一个共同的膀胱和尿道。泌尿系统的主要功能是通过管理血液的水和电解质平衡来维持身体的稳态。肾脏过滤血液以控制电解质浓度和酸碱平衡, 并产生尿液排出过量的水和废物, 包括溶质和代谢物。然后, 尿液通过输尿管从肾脏的肾盂转移到 bladderin 的单向的方式, 它是储存和最终消除通过尿道1。
输尿管是来自肾导管的直管。在小鼠胚胎日 10.5 (E10.5) 的肾导管萌芽后, 输尿管柄拉长并分化成一种称为膀胱的多层结构, 在小鼠 E12.5 和 E16.5 之间是不透水的。输尿管柄周围的间充质细胞也被分为三层, 包括内基质细胞、中层粗平滑肌细胞和外外成纤维细胞。输尿管蠕动波起始于肾盂, 通过输尿管壁的平滑肌层传播至膀胱以传输尿液2,3, 它是在鼠标1中从 E16.5 开始生成的。
先天性异常的肾脏和泌尿道 (CAKUT) 是最常见的遗传性疾病, 目前在大约1% 的人类胎儿1,4, 并组成的各种表型包括肾积水和 us。肾脏和输尿管的尿液异常积累导致积水肾和 us 的形成。肾积水和/或 us 形成的一个原因是尿路梗阻。肾盂结肠梗阻 (UPJO) 是由于在近端输尿管和肾盂之间的堵塞造成的尿流异常, 导致肾积水和近端 us 狭窄与角度或持续折叠5, 6。此外, 远端输尿管的异位插入膀胱壁或生殖道称为输尿管结肠梗阻 (UVJO)。UVJO 还可诱发肾积水和 hydromegaureter 形成7,8。另外的输尿管结 (UVJ) 缺陷是膀胱回流 (VUR)。VUR 的特点是逆行尿流从膀胱向肾脏在 UVJ。与 UVJO 相比, 围产期胚胎与 VUR 不明显显示肿胀积水肾或严重 us 表型9。
在实验小鼠, 尿液流动可以通过注射染料, 如亚甲蓝, 进入肾盂9。injectedmethylene 蓝溶液将从肾盂经输尿管进入膀胱, 追踪尿液轨迹。肾积水可以通过在肾脏中的染料的膨胀来识别。UPJO 可被检测为堵塞的染料流在近端输尿管与肿胀肾盂5。经加宽的直径所示的输尿管的任何扩张都显示了 us 的一个例子。最后, 染料堆积在膀胱壁或在生殖道的站点表明 UVJO 与膨胀的积水肾脏和扩张的 hydromegaureter7,10。为了检测 VUR, 染料溶液注入膀胱, 随后的逆行流在肾脏的9中被监测。
在这里, 提出了一个协议的亚甲蓝染料注射到肾盂的围产期胚胎。这项协议允许追踪尿液从肾盂通过输尿管和进入膀胱, 并验证潜在的泌尿道交界处的障碍, 如 UPJO 或 UVJO。

<table width="786">
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		<tr height="21">
			<td height="21" style="width:295px;height:21px;">Methylene blue</td>
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assessing urinary tract junction obstruction defects by methylene blue dye injection

尿路结肠梗阻是先天性异常, 诱发肾积水和 us。小鼠泌尿道结肠梗阻的缺陷可以通过跟踪在泌尿系统内的亚甲基蓝染料流动来评估。将亚甲蓝染料注入围产期胚胎肾脏的肾盂中, 在应用静水压后, 通过输尿管肾脏的肾盂和膀胱腔内的染料流进行监测。在正常围产期泌尿道的膀胱腔内, 染料堆积明显, 但在肾盂与输尿管异常的终点之间, 如果出现尿路梗阻, 则会受到限制。该方法可促进尿路结肠梗阻的确诊和肾积水和 us 的可视化。这份手稿描述了一个协议的亚甲蓝染料注射液进入肾盂, 确认泌尿道交界处的障碍。

肾脏和下泌尿系统位于背部的大多数其他内脏器官, 如肝脏和肠道。取出这些其他内脏器官后, 可看到一对肾脏和输尿管和一个膀胱 (图 1A)。成功解剖后, 注入肾盂的染料将通过输尿管从肾脏流向膀胱。肾盂是肾中输尿管的漏斗状扩张的近端部分。因此, 染料注射点附近的可见近端输尿管与肾脏连接, 用黑色箭头表示 (图 1B-1...

Watch this Scientific Journal Video about Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection at JoVE.com

Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

亚甲蓝染料注射入肾盂有助于评估尿路结肠梗阻的缺陷, 在小鼠胚胎泌尿道发育。在这里, 一个协议的亚甲蓝染料注射到肾盂是描述。

亚甲蓝染料注射液评价泌尿道结肠梗阻的缺陷

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction ...

assessing-urinary-tract-junction-obstruction-defects-methylene-blue

Research

JoVE Journal

Biology

15.5K 次观看.  National Institutes of Health. 亚甲蓝染料注射入肾盂有助于评估尿路结肠梗阻的缺陷, 在小鼠胚胎泌尿道发育。在这里, 一个协议的亚甲蓝染料注射到肾盂是描述。

视频: Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

线虫神经肌肉交界处的解剖和录制

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

科研

生物学

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

斑马鱼脑室注射液

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

Ferumoxides植入人类间质干细胞在软骨缺损的标记

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi的干扰双链RNA注射到果蝇胚胎

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

小肠部分梗阻的小鼠模型

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

一种碘-黄色荧光荧光蛋白-缝隙--细胞间通信方法

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

用于评估小鼠生殖道自发性的方法和用于数据分析的基于 MATLAB 的 Uterus 运动跟踪算法

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...