这项工作得到了中央大学基础研究基金 (020814380018, 020814380077), 中国奖学金理事会 (CSC, 201606195024 号), 江苏省自然科学基金 (BK20160138) 和重点项目的支持。由南京医科大学科技发展基金会 (2014NJMUZD053) 资助。

本研究采用三月大、18月大的大鼠。所有动物都是按照 NIH 的护理和使用实验动物的指导方针处理的。涉及动物问题的程序得到当地机构动物照料和道德委员会的批准, 努力尽量减少动物的痛苦。这些议定书是由南京理工大学 (中国南京) 机构动物保育和使用委员会 (IACUC) 批准的。
在 ICP 记录前, 根据其年龄和早期表现, 将大鼠分为两组: 青年正常组 (YN 组) 和老年勃起功能障碍组 (AE 组)10。
1. 手术前准备
<ol><li>手动为 ICP 记录制作一对双极性电极 (图 1)。稍微弯曲电极的两端, 并将两个电极之间的距离调整为1-2 毫米宽, 如图 1A所示。</li>
	<li>使用两个鳄鱼钳 (图 1A, B) 将电极连接到刺激器。</li>
	<li>
装配导管系统:首先, 将皮下注射的23G 针连接到带有油管的3路旋塞阀, 然后将旋塞阀连接到压力传感器。接下来, 在旋塞阀的第三端附上一个10毫升的注射器, 以提供肝素生理盐水。</li>
	<li>在用肝素盐 (200 U/毫升) 填充整个系统后, 仔细检查是否有渗漏。然后将3路旋塞阀关闭注射器通道或压力传感器通道(图 1C)。</li>
	<li>提起针20厘米的木垫水平。然后将压力记录系统校准到20厘米 H2O。之后, 移动针的高度, 以验证记录系统的准确性。重复校准, 直到确认准确性。</li>
	<li>将老鼠从动物设施转移到手术室, 让他们习惯于手术室至少30分钟。</li>
	<li>在手术前用70% 乙醇喷洒蒸压设备</li>
</ol>2. 手术程序
<ol><li>麻醉大鼠腹腔注射戊巴比妥钠, 剂量为45毫克/千克体重, 并等待5-10 分钟. 捏脚趾以确认适当的麻醉。</li>
	<li>用电动剃须刀剃去腹部和颈部的皮毛, 然后把老鼠放在暖气垫上。</li>
	<li>用10% 聚维酮碘溶液浸泡棉球, 然后用70% 只乙醇浸泡棉球擦拭手术区。同时, 应用眼科软膏防止眼部干燥。</li>
	<li>Catheterize 左颈动脉。<ol>
<li>用镊子抓住颈部的皮肤, 在颈部中部做水平切口。切开肌肉, 仔细暴露左颈动脉, 并隔离血管的5毫米部分。</li>
		<li>用镊子仔细地将颈动脉与迷走神经分开, 在颈动脉下画一条丝缝线, 并在血管的尾端放一条松散的领带, 然后在血管的颅骨末端再做一个紧的结。</li>
		<li>Caudally 钳夹在缝合线上方的牛头犬钳, 以阻止血液流动。</li>
		<li>用显微外科剪刀在血管上做切口, 在微解剖钩和镊子的协助下, 将动脉导管插入心脏。</li>
		<li>把松动的尾鳍绑在导管上以确保它的安全。取出牛头犬钳以恢复血液流动。</li>
	</ol>
</li>
	<li>
隔离 CN 并放置电极
	<ol>
<li>用钳子抬起腹部的皮肤和肌肉。用解剖剪刀, 切开下腹部到小弟弟做中线切开。</li>
		<li>用拭子轻轻地将肠道推入腹腔上部。</li>
		<li>用一副镊子抓住膀胱, 从腹腔取出膀胱。暴露前列腺的腹叶, 位于尿道的腹部。</li>
		<li>拔出前列腺、精囊和输精管的腹叶, 露出前列腺的背叶。找出输精管和前列腺的粘连点。</li>
		<li>分离前列腺和输精管之间的空间。仔细暴露的纤维胶囊, 这是位于后到连接点的前列腺和输精管。然后找到主要的骨盆神经节 (图 2)。 注: 在前列腺表面可以看到主要的盆腔神经节和海绵状神经。</li>
		<li>小心干燥 CN 区域与无菌拭子。用两极电极仔细隔离并钩住右海绵状神经。</li>
	</ol>
</li>
	<li>
Catheterize 左下肢
	<ol>
<li>用解剖剪刀在小弟弟的皮肤上切开一个小切口, 然后仔细掀的小弟弟的皮肤。</li>
		<li>解剖纹状的小弟弟肌组织。找到耻骨骨的上部分支。</li>
		<li>暴露 bulbospongiosus 的肌肉, 包括 spongious 灯泡。</li>
		<li>用弯钳将 bulbospongiosus 肌从 ischiocavernosus 肌中分离出来。</li>
		<li>小心地将 ischiocavernosus 肌肉与弯曲钳隔离, 然后切开 ischiocavernosus 肌以暴露小腿海绵的白色膜。</li>
		<li>遵循小腿语料库海绵的解剖方向, 小心地将针插入小腿语料库海绵通过白色的膜。 注意: 这是成功导尿的关键步骤。如果针插入正确, 少量的血气盐水可以被注射, 并且应该观察轻微的小弟弟膨胀。</li>
		<li>小心释放针头, 避免任何滑动的针头或中断连接管。检查是否有渗漏。</li>
	</ol>
</li>
</ol>3. 刺激 CN
<ol><li>打开压力信号记录软件程序, 启动压力信号记录。</li>
	<li>设置刺激的参数:15 赫兹, 脉冲宽度为5毫秒, 5 伏, 和六十年代的持续时间。以15赫兹的频率刺激 CN, 脉冲宽度为5毫秒。 注: 在应用电刺激时, 可观察到 ICP 的急剧上升。</li>
	<li>让刺激之间有30分钟的休息时间。每只动物的连续刺激的最大值是三倍。</li>
</ol>4. 结束程序
<ol><li>记录后, 通过注射过量的戊巴比妥钠剂量150毫克/千克体重来管理安乐死。通过检查动脉压力来证实老鼠的死亡。取出老鼠, 清理手术工具。</li>
</ol>5. 数据分析
<ol><li>保存并导出软件中的数据。反应通常表示为 ICP 与全身平均动脉压的比值 (图)。计算了峰值比值/MAP 对勃起功能的评价。</li>
	<li>从至少五只老鼠那里采集数据, 并用统计软件进行分析。当p < 0.05 使用学生的 t 测试时, 差异被认为具有统计学意义。</li>
</ol>

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作者没有什么可透露的

作为勃起功能的直接措施, ICP 是一种可靠的方法14。它允许获得关于基 icp 备、峰值比较、高原 icp、安装时间和消肿时间、响应时间、等的数据。除这些直接测量参数外, 还有一些其它指标参数: (1) "T80", 达到80% 峰值的时间;(2) "D20", 时间减少到20% 峰值 ICP;(3) "ΔT80", T80 压力增加率 (每秒);(4) "ΔD20", 在 D20 的压力降低率 (每秒)。这些参数允许量化每个阶段和 ICP 反应的质量, 这可...

ED 被定义为无法达到或保持勃起, 并已成为常见的男性性障碍1。实验动物被使用并且提供可再生的模型调查勃起功能2。长期以来, 一些较大的动物模型被用于调查勃起功能3,4,5。虽然啮齿动物比其他动物相对较小, 但它们也被用来研究男性勃起功能障碍, 因为它表现出了几个优点6。首先, 人类的形态和功能性特征在啮齿类动物中概括。第二, 与 ED 研究中使用的大型动物相比, 啮齿动物更经济的购买, 房子和维护。第三, 转基因啮齿动物模型在重现性和后续行为以及神经研究中提供了优势。因此, 啮齿类动物很快就成为了研究男性勃起功能障碍的主要方法。
啮齿动物模型得益于纯遗传背景和一致的文化条件, 提供了一致的可重现数据5,6,7,8。在许多与勃起功能相关的研究中, 阿扑吗啡的勃起反应试验和电刺激诱发的 ICP 反应试验是最广泛使用的方法, 可以可靠地反映勃起函数9,10,11,12。由希顿et 等开发的蛋白诱导的勃起功能测试。13是一种生物检测方法, 它利用阿扑吗啡对大鼠进行勃起和打呵欠的现象。作为一种简便、无创、稳定的生物检测方法, 对勃起功能进行评价, 在许多研究中广泛应用了该蛋白诱导的勃起功能检测。然而, 这项化验并不能充分反映勃起的质量或血液流量的动态变化, 与阳痿的反应14。ICP 测量最初是由昆兰et 等人开发的。15. 在该方法中, 将导管放入颈动脉以测量全身血压, 另一导管插入小腿语料库海绵以记录 ICP。在 ICP 记录之前或期间, 主要骨盆神经节 (加仑) 或 CN 的血管活性剂和/或电场刺激经常被给予老鼠14。这种检测方法是评估 ED 治疗和药物的可靠工具, 在未来的6中很有可能被用作重要的评估手段。
与易于执行的蛋白能诱发的勃起功能测试相比, 成功的 ICP 记录需要注意细节、练习和坚持操作方法。因此, 在这里, 我们提供了如何执行 ICP 记录的详细说明。

<table border="0" cellpadding="0" cellspacing="0" style="width:1099px;" width="1098">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Animal</td>
			<td style="width:289px;"></td>
			<td style="width:336px;"></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rats</td>
			<td></td>
			<td></td>
			<td>Strain: Sprague-Dawley 
			Age: 2-3 month</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rats</td>
			<td></td>
			<td></td>
			<td>Strain: Sprague-Dawley 
			Age: 15-18 month</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagents</td>
			<td></td>
			<td></td>
			<td>formula</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Saline</td>
			<td style="width:289px;">Sigma-Aldrich, S7653</td>
			<td style="width:336px;"></td>
			<td style="width:345px;">dissolve 8.5 gram sodium chloride in distilled water</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:128px;">Pentobarbital sodium solution</td>
			<td style="width:289px;">Sigma-Aldrich, P3761</td>
			<td style="width:336px;"></td>
			<td style="width:345px;">dissolve 1 gram in 100 ml saline</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:128px;">Povidone-iodine</td>
			<td>BTP Pharmaceutical Co. Limited</td>
			<td></td>
			<td style="width:345px;">10% (V/V)</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:128px;">Ethanol</td>
			<td style="width:289px;">China National Pharmaceutical Group Corporation (SINOPHARM)</td>
			<td style="width:336px;"></td>
			<td style="width:345px;">70% (V/V)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Heparin</td>
			<td style="width:289px;">Sigma-Aldrich, H3149</td>
			<td style="width:336px;"></td>
			<td style="width:345px;">dissolve 20000U heparin in 100 ml saline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Materials</td>
			<td></td>
			<td colspan="2"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:128px;">Hypodermic needle</td>
			<td style="width:289px;">Shandong Weigao Group Medical Polymer Co., Ltd.&#160;</td>
			<td>23G</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:128px;">Syringe</td>
			<td style="width:289px;">Shandong Weigao Group Medical Polymer Co., Ltd</td>
			<td>10 ml</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Three-way stopcock</td>
			<td style="width:289px;">Chengdu Instrument factory</td>
			<td>TSK 01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Electrode</td>
			<td style="width:289px;">Chengdu Instrument factory</td>
			<td>JST-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Catheter tube</td>
			<td style="width:289px;">Chengdu Instrument factory</td>
			<td>PE-10, PE-50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Operating scissors</td>
			<td>Shanghai operation equipment factory</td>
			<td>J22010, J22020</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:128px;">Ophthalmic operating scissors</td>
			<td>Shanghai operation equipment factory</td>
			<td>Y00010, Y00020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Ophthalmic forceps</td>
			<td>Shanghai operation equipment factory</td>
			<td>JD1010, JD1020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">MicroScissors</td>
			<td>World Precision Instruments</td>
			<td>WAA260</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:128px;">silk suture</td>
			<td style="width:289px;">Shandong Weigao Group Medical Polymer Co., Ltd.&#160;</td>
			<td>5-0</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td colspan="2"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:128px;">Stimulator</td>
			<td style="width:289px;">Nanjing medease science and technology co. ltd (model 4C501H)</td>
			<td style="width:336px;"></td>
			<td style="width:345px;">15 Hz, 5 ms pulse, 5 V, 60 s duration and 5 minutes interval</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:128px;">Multichannel signal collection processing system</td>
			<td style="width:289px;">Nanjing medease science and technology co. ltd (model 4C501H)</td>
			<td style="width:336px;"></td>
			<td>Blood pressure model</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:128px;">Pressure transducer</td>
			<td style="width:289px;">Beijing Xin Hang Xing Ye Technology Trading Company Limited (model YP100)</td>
			<td style="width:336px;"></td>
			<td>40KPa</td>
		</tr>
	</tbody>
</table>

intracavernosal pressure recording to evaluate erectile function in rodents

勃起功能障碍 (ED) 被定义为不能达到或保持勃起的小弟弟, 这已经成为一个普遍的男性性障碍。啮齿类动物被许多研究用来研究勃起功能的生理学/病理学。通过测量 intracavernosal 压力 (ICP) 可以评价啮齿动物的勃起功能。在实际应用中, 可以通过电刺激海绵神经 (CNs) 来监测 ICP。应用颈动脉压 (平均动脉压) 作为 ICP 的参考。利用 icp 记录协议, 可以从 icp 反应曲线测量勃起功能的许多关键参数。ICP 测量比阿扑吗啡诱导的勃起试验提供更多的信息, 而且比海绵的人的小弟弟的遥测监测更便宜, 这使得这种方法最受欢迎的一个评价阳痿的功能。然而, 与易于执行的蛋白能诱导勃起功能测试相比, 成功的 ICP 记录需要注意细节, 实践和坚持的操作方法。在这项工作中, 介绍了在大鼠的 ICP 记录, 以有效补充程序。

许多研究表明, 老年男性勃起功能障碍正成为一个普遍的问题。然而, 在管理衰老相关的教育16方面, 医疗治疗受到限制。在衰老相关的啮齿动物模型中, 对老龄大鼠的勃起功能进行了多种治疗。正如我们前面所介绍的那样, ICP 记录试验可用于将 ED 动物与实验动物的总人口区分开来, 这对于量化潜在治疗或药物对勃起功能的影响也很有价值。
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Watch this Scientific Journal Video about Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents at JoVE.com

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Intracavernosal 压力记录对啮齿动物勃起功能的评价

Intracavernosal 压力记录 (ICP) 是评价实验动物勃起功能的重要方法。在这里, 通过置管下肢的小弟弟, 然后电刺激大鼠的海绵神经, 证明了比较 ICP 的记录过程的详细协议。

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. ...

intracavernosal-pressure-recording-to-evaluate-erectile-function

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.1K 次观看.  Nanjing University. Intracavernosal 压力记录 (ICP) 是评价实验动物勃起功能的重要方法。在这里, 通过置管下肢的小弟弟, 然后电刺激大鼠的海绵神经, 证明了比较 ICP 的记录过程的详细协议。

视频: Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

在啮齿类动物的存活立体定向手术

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

科研

生物学

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

改进的膜片钳记录的压力抛光移液器

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

使用卷压力记录，一条尾巴袖套法小鼠的血压测量

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

探讨动脉平滑肌的KV7钾离子通道功能使用膜片钳电生理和压力Myography

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

评估抗小鼠动脉功能使用压力Myography

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

在收缩力测量评估肌肉功能在斑马鱼幼虫

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

基于光二极管的光学成像，用于记录非转基因无脊椎动物的单神经元分辨率的网络动态

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.