Supported by NIH award R01 DK093525 (GFG). We greatly appreciate the scientific review and grammatical editing of the manuscript by Dr. Amber Shaffer (University of Pittsburgh) and thank Michael Burcham for assistance in preparation of figures.

注：该协议是审查和批准匹兹堡机构动物护理和使用委员会的大学。 1.准备修改克雷布斯解决方案和测试药物的等分<ol><li>使6升的改良的Krebs溶液包含（以mM计）：117.9氯化钠，氯化钾4.7，25的NaHCO 3，1.3的NaH 2 PO 4，1.2 MgSO 4干燥，2.5氯化钙2，11.1 D-葡萄糖，2-丁酸钠，乙酸钠20 ，0.004硝苯地平（阻止自发性肌肉收缩），以及0.003吲哚美辛（阻断内源性合成前列腺素）。用冰冷和温暖的克雷布斯解决方案的组织剥离和单纤维记录，分别为。 </li><li>准备任何化学溶液（ 如 IS，辣椒素，ATP）在分装时所需的浓度。 </li></ol> 2.解剖结直肠神经组织<ol><li>麻醉和安乐死雄性小鼠（6 - 8周龄，20 - 30克）的在CO 2室中的流速即位移10 -每分钟的腔室容积，直至小鼠的30％停止呼吸完全没有胸部运动所指示的。 </li><li>紧接安乐死，抽血通过切割打开胸腔，射孔右心房，并浸渍鼠标胎体在充足体积的冰冷的Krebs（〜500毫升）（4℃）溶液鼓入卡波金（95％O 2的，5％的CO 2）。 </li><li>小心地取出所有的内脏，但结肠和盆腔器官。断面跨T12脊髓节段鼠标的一半，略高于横膈膜和尾部半转移到含有冰冷的解剖腔，起泡克雷布斯解决方案。 </li><li>在立体显微镜下，通过横断他们的路口尿道取出膀胱和生殖器官，并删除降/腹主动脉，直至其分叉成髂总动脉。释放PN或从他们surroundin的LSN克组织钝性分离，并按照从髂嵴，直至其腹入口点L6和S1椎列（PN）或T13和L1椎列（LSN）以外的神经。 </li><li>切开耻骨联合和左，右髋关节，去除骨骼。仔细自由无论是PN或LSN从接近结直肠，直到那里的神经进入脊柱附着的肌肉和结缔组织。 </li><li>仔细切除髂骨，露出远端结肠直肠。解剖出远端结肠直肠与连续所附PN或LSN。 </li><li>转移带贴在组织腔的浴室中的神经结直肠。通过进一步解剖除去过量结缔组织，并纵向沿着反肠系膜边界打开结肠直肠。 </li><li>与粘膜的一面朝上，PIN结直肠的靠近记录室中的肠系膜边缘进入室使用的聚硅氧烷基r和附加到连接到一个力致动器挂钩的耙结肠直肠的系膜长度（示于图1和拍摄在图2A）。 </li><li>延长PN或LSN到记录室，它是由一鼠标孔和栅极连接到该浴室中。轻轻地将神经干到在录音室中，这为神经坚持亲水表面的小玻璃镜面。 Superfuse洗澡舱用温水（30 - 32°C），含氧克雷布斯解决方案，填补与矿物油的录音室中。 </li></ol> 3.单纤维记录和感受野的本地化<ol><li>小心剥离回从PN或LSN神经外膜（神经鞘）的体视显微镜，在高放大倍率（50 - 60X）下。用细镊子，挑逗神经干到5 - 〜100微米厚度的8神经束。 </li><li>将铂 - 铱参考电极与在所述组织腔Krebs溶液接触。依次将各个神经束到由相同材料制成的记录电极。 </li><li>使用软画笔轻轻抚摸上下结表面，以唤起从大肠传入的AP。找到神经束（S），通过检测AP（动作电位）录音支配结肠。 注：PN和LSN也支配膀胱等盆腔器官。 </li><li>使用一对为30g针尖的神经束，以进一步分成10微米厚的556肌束长丝和放置一个单独的长丝到记录电极。 </li><li>将圆尖同心电极垂直于粘膜表面，以电激发传入神经末梢在阈上刺激强度（10毫安幅度，0.5毫秒持续时间@ 0.3赫兹），其产生的电流扩散一〜半径2毫米。将电极系统（1〜1.5毫米间距）沿着所述扁平大肠的长度和宽度来本地化接受的结局。 </li><li>当传入结局被激发，调整电极位置精确定位的激活（感受野，RF）的一个需要的最小刺激强度（刺激阈值）的位点。丢弃的结局与刺激的阈值&gt;3毫安19。 </li><li>计算刺激伪迹和动作电位的发生之间的传导速度（CV）从1）在感受野（RF）的刺激电极和记录位点和2）的导通延迟（ 例如， 图2B之间的距离） 。 CV（米/秒）=距离（mm）/传导延迟（毫秒）。 </li></ol>机械敏感性结传入放电4.功能分类<ol><li>定位一个射频电刺激后，取以下三个机械刺激到RF： <ol><li>通过按压CALIBRA的前端进行探测刺激特德·冯·弗雷，如尼龙单丝（0.4和1克力）朝垂直的扁平结直肠的RF。 </li><li>轻轻抚摸着大肠黏膜用细尼龙丝股（10毫克力）产生的RF小面剪应力进行抚摸刺激。 </li><li>用计算机控制的力致动器，它提供了一个倾斜的拉伸力进行周向拉伸（0 - 170 MN在5分钟/秒）沿大肠经由在步骤2.8中所述钩的耙抗肠系膜边缘圆周方向。 </li></ol></li><li>分类传入为浆膜（只响应钝探测），粘膜（回应粘膜抚摸和钝探测），肌肉（回应周向拉伸和钝探测）肌肉/粘膜（回应周向拉伸，粘膜抚摸和钝探测），或MIA（未响应于任何三个机械刺激的）。 </li><li>对于肠系膜传入（仅在该LSN支配），很难通过电刺激选择性激活，找到自己接受的结局由机械轻抚/探测肠系膜。 </li><li>对于拉伸敏感传入（肌肉和肌肉粘膜），确定响应的阈值，其被定义为，唤起的第一个AP期间倾斜拉伸的力。 </li><li>对于浆膜传入，记录他们的反应，上升点状探测的感受野由计算机控制的力致动器驱动的水平。 </li></ol>感受结尾5.化学应用/调制<ol><li>记录基线响应机械刺激（ 即响应斜坡舒展，点状探测，或粘膜行程）。 </li><li>外套一块管（黄铜或不锈钢，高10毫米和4×4 平方毫米的方形或4 -直径5mm）的底部边缘与凡士林，并将其放置在对结直肠感受野。 </li><li>拆除克雷布斯溶液的管内，并露出接受结束3 - 5分钟到170微升含有化学（S）以被测试的溶液中。 </li><li>监测过程中的化学应用传入的响应（有些传入的化疗敏感）。 </li><li>除去化学溶液和油管终止化学的作用。在4 - 6分钟，测试传入响应于相同的机械刺激如在基线响应。 </li><li>后洗出足够的时间（&gt; 15分钟）再重新应用机械刺激。 </li></ol> 6.记录和判别AP尖峰<ol><li>数字化记录在从轴突在20千赫的电信号并将数据保存到计算机。通过音频监视器监控上线的信号。 </li><li>分析AP尖峰离线和歧视基于个人尖峰波形25的主成分分析单个单位。 注意：一个记录不应包含更多次一个2轻松地辨别现役部队。 </li></ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+visceral+hypersensitivity+by+glial+cell+line-derived+neurotrophic+factor+family+receptor+&#945;-3+in+colorectal+afferents.">Modulation of visceral hypersensitivity by glial cell line-derived neurotrophic factor family receptor &#945;-3 in colorectal afferents.</a> Am J Physiol Gastrointest Liver Physiol. 300, G418-G424 (2011).</li></ol>

The authors have nothing to disclose.

这里所描述的在体外结直肠神经制剂已被证明是一个有效的方法来研究各个结传入，它很好的补充了对内脏感觉神经元等非功能性的方法（ 例如，细胞，分子和组织学研究）的神经编码功能（见综述27的详细信息）。促进伤害感受和长期结肠超敏反应神经机制已经揭示和药理操作已经执行了具有可以减轻内脏痛显示目标。成功实施本制剂的相关联的以下要点讨论如下：1）减?...

疼痛和过敏症是患者的主要抱怨从功能性胃肠疾病，包括肠易激综合征（IBS），在没有明显的病理生物学原因或组织损伤中存在的痛苦。例如，IBS患者表现出过敏症，包括在正常的肠功能提高反应直肠球囊扩张和增加的敏感性，以及体细胞介超敏反应（ 即，压痛到腹部的触诊）1。因为靶向结肠传入已被证明是有效的，以减轻疼痛和过敏症中IBS患者（ 例如，局部麻醉药2,3-直肠内滴注;鸟苷酸环化酶-C的利那洛肽激动剂4-6的口服摄取），改进的理解结直肠传入神经神经支配是很重要的。 内脏传入，包括结肠传入，能够应对化学/营养和热模式（ 如 7-9）的。然而，内脏传入响应于机械性刺激（ 即，机械敏感传入神经 ​​）一直是最彻底的研究，因为机械性刺激（ 例如，管腔腹胀，拉伸）是那些通常对感觉的，包括不适和疼痛10-16产生。此外，内脏也支配通过机械敏感传入神经 ​​（MIA中），通常被称为无声或睡眠伤害感受器17。在正常生理条件下，MIA中不回应机械性刺激或有非常高的反应阈值18，但可以成为积极的，并获得mechanosensitivity在病理情况下，有助于过敏。 使用体外准备和这里描述的协议，我们开发并采用电刺激策略，海<html> RCH为接受的结局，允许公正的鉴定两种机械敏感性和MIA的结局在结直肠19。大肠支配从腰内脏（LSN）和骨盆神经（PN）的途径获得，并且包括可以被分类成五个机械敏感类（浆膜，粘膜，肌肉，肌肉粘膜，肠系膜）和一个MIA类20大肠传入。使用这种在体外制备，我们发现，大肠癌MIA中取得mechanosensitivity（致敏）以下的感受域的短暂暴露于炎性汤（IS），这在LSN通路敏化在PN通路MIA中的71％和MIA中的23％的19。我们也记录MIA中的长期敏（28天）的长期行为内脏过敏的情况下（ 即在结肠内接受治疗与酵母多糖21或2,4,6-三硝基苯磺酸小鼠（TNBS）22） 。 jove_content“&gt;其中机械敏感性传入，肌肉发达，肌肉，黏膜传入是唯一的类tonically编码结直肠周向拉伸（ 即是拉伸敏感）和subserve性结直肠扩张23,24的编码，利用计算机控制力致动器，我们应用一个标准的，同质的，且可重复倾斜伸展的扁平大肠组织的圆周方向和进一步分类拉伸敏感传入神经 ​​作为低阈值和高阈值23，此外，拉伸的致敏的时间过程经过酵母多糖结肠内21或22 TNBS治疗敏感的传入对应于起病，持续性，和/或行为内脏敏感性的恢复，提示舒展敏感结传入内脏疼痛和感觉过敏的作用。

<table border="0" cellpadding="0" cellspacing="0" width="1173">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Name of Material/ Equipment</td>
			<td style="width:291px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:372px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica MZ16 stereo microscope&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica IC D camera</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amplifier</td>
			<td>World Precision instruments, Inc.</td>
			<td>SYS-DAM80&#160;</td>
			<td>Low-noise differential amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Two-compartment tissue chamber</td>
			<td style="width:291px;">Custom made</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Power1401</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;">Power1401</td>
			<td>Data acquisition, analog signal input/out</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spike2 v5.02</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;"></td>
			<td>Software package that works with the Power1401</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Audio monitor</td>
			<td>Natus</td>
			<td style="width:137px;">Am 8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Square pulse stimulator</td>
			<td>Natus</td>
			<td style="width:137px;">S48</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Photoelectric isolation unit</td>
			<td>Natus</td>
			<td style="width:137px;">PSIU6</td>
			<td>Stimulus isolation to reduce noise</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Concentric bipolar microelectrode</td>
			<td style="width:291px;">FHC Inc.</td>
			<td style="width:137px;">CBFFG75</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual-mode lever system</td>
			<td>Aurora Scientific Inc.</td>
			<td style="width:137px;">Series 300C&#160;</td>
			<td>To deliver mechanical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>forceps with fine tips</td>
		</tr>
	</tbody>
</table>

in vitro functional characterization of mouse colorectal afferent endings

详细此视频演示了使用鼠标结直肠神经制剂体外单纤维电生理记录协议。该方法允许公正的鉴定和个人结传入的功能特性。传播动作电位（APS），从一个或几个传入神经 ​​起源的细胞外记录（ 即，单纤维）感受域（RFS）在结直肠从逗神经纤维束状制成。结直肠与任何骨盆（PN）或连接并打开纵向腰内脏（LSN）神经去除。该组织被放置在记录室中，钉扎平并灌注充氧的Krebs溶液。焦电刺激被用于定位大肠癌的传入神经末梢，它们由三个不同的机械刺激进一步测试（钝探测，粘膜划线和周向拉伸）到传入功能分类成五个mechanosensi略去类。结局回应没有这些机械的刺激被归类为机械不敏感的传入（MIA中）。两个机械敏感和MIA中可评估致敏（ 即，增强的响应，减小阈值，和/或获取mechanosensitivity的）通过射频对化学品的局部暴露（ 例如，炎性汤（IS），辣椒素，三磷酸腺苷（ATP））。我们描述的设备和结直肠神经录音准备，收获结直肠附加PN或LSN，在结直肠，单纤维从神经束记录射频识别的，和化学品的本地化应用的RF。此外，制备和应用的标准化机械刺激的挑战进行了讨论。

设置示于图1，它包括一个特制的组织腔，其容纳在硅氧烷衬里的浴室并在一个连续的矿物油填充室中的附神经结直肠。两室室从丙烯酸塑料由CNC机床的固体块加工;两个隔室的底部，随后衬有牢固的硅氧烷，以允许容易销的大肠癌组织中向下。从梳理神经束外的AP使用低噪声，电池供电的差分放大器具有高共模抑制比（CMRR〜60 dB为单位）被记录。放大器的增益被设定为x10,000和带过滤器...

Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

在小鼠大肠传入结尾的体外功能研究

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

in-vitro-functional-characterization-mouse-colorectal-afferent

Research

JoVE Journal

Medicine

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

9.5K 次观看.  University of Pittsburgh. This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation. 

视频: In vitro Functional Characterization of Mouse Colorectal Afferent Endings

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

利用人类纤维原细胞线粒体表型的监测领域的帕金森氏病

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

科研

医学

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

在体外化培养基层小鼠结肠肿瘤

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

建立和UTI和CAUTI表征在小鼠模型

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

在横向主动脉缩窄的小鼠模型和心脏形态学评估功能改变通过超声心动图成像

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

纵<em&gt;在体内</em&gt;的Cerebrovasculature影像：相关性疾病CNS

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

制备和<em&gt;体外</em&gt;磁化的表征的miR-改性内皮细胞

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

小鼠致耐受性树突状细胞的发育及功能表征

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

内源性表达人RYR1变异体的功能表征

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

Occlusion of the Great and Small Saphenous Vein Using Copolymeric Glue Based on N-Butyl Cyanoacrylate and Methacryloxy Sulfolane

We present the preliminary results of a longitudinal observational study aimed at evaluating the effectiveness and safety at different short- and long-term follow-ups of vascular occlusion of the great saphenous vein (GSV) and small saphenous vein (SSV) affected by severe pathological reflux, using an innovative modified cyanoacrylate surgical glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS). Ninety patients, prospectively recruited for 1 year, underwent the study with EcoColor-Doppler (ECD) to evaluate the maximum diameters of the GSV and SSV in the orthostatic position and the reflux time (RT). An RT greater than 0.5 s was considered pathologic. Clinical, etiology, anatomy, and pathophysiology (CEAP) assessment was used for the complete evaluation of each patient in the study. All the patients were treated by NBCA+MS glue to obtain vein occlusion and observed before treatment (baseline; T0), within 6 h after treatment (T1), 1 month after treatment (T2), 3 months after treatment (T3), 6 months after treatment (T4), and 1 year after treatment (T5). Chi-square (&#967;) analysis was performed to evaluate the effectiveness and safety of the treatment. All the patients participated in the entire duration of the study. Complete occlusion was maintained in 100% of patients at T1, 98.9% at T2 and T3, and 97.8% at T4 and T5 (p &lt; 0.001). None of the patients suffered from post-surgical thrombosis. No blue hyperpigmentation, or paresthesia was observed during the entire observation period. Immediately after treatment, 7.7% of patients needed painkillers; 1 week after treatment, 100% of patients returned to normal life. Vascular occlusion of the great or small saphenous vein using NBCA+MS glue is a safe procedure with persistent benefits after a 1 year follow-up. This procedure can be performed with local anesthesia, allowing a quick return to normal life. Thanks to its low invasiveness, the treatment is not painful.

Here, we present a protocol to treat the great saphenous vein (GSV) and the small saphenous vein (SSV) affected by severe pathological reflux, using an original sclerosing and embolizing cyanoacrylate-based glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS).