Name: microelectrode impalement method to record membrane potential from a cannulated middle cerebral artery
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这项工作部分得到了UMMC的校内支持研究计划(IRSP)的资助,AHA科学家发展赠款(13SDG14000006)授予了Mallikarjuna R. Pabbidi。

雄性大鼠被安置在UMMC的动物护理设施中,该设施得到实验室动物护理评估和认证协会(AAALAC)的批准。在整个研究中,动物可以免费获得食物和水。动物在受控环境中保持温度为24~2°C,湿度水平为60~80%,光/暗循环为12小时。所有协议均获得UMMC动物护理和使用委员会的批准。
1. 设备准备
<ol><li>将双通道差分仪电表放大器(参见材料表)靠近容器室和所需位置。</li>
	<li>使用...

<ol>
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A., 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=Fundamental+increase+in+pressure-dependent+constriction+of+brain+parenchymal+arterioles+from+subarachnoid+hemorrhage+model+rats+due+to+membrane+depolarization.">Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization.</a> American Journal of Physiology-Heart and Circulatory Physiology. 300 (3), H803-H812 (2011).</li><li>Yvonder Weid, P., Lee, S., Imtiaz, M. S., Zawieja, D. C., Davis, M. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+membrane+properties+in+vascular+muscle+cells+from+various+vascular+beds.">Heterogeneity of membrane properties in vascular muscle cells from various vascular beds.</a> Federation Proceedings. 42 (2), 253-256 (1983).</li><li>Cogolludo, A., 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=Serotonin+inhibits+voltage-gated+K++currents+in+pulmonary+artery+smooth+muscle+cells:+role+of+5-HT2A+receptors,+caveolin-1,+and+KV1.5+channel+internalization.">Serotonin inhibits voltage-gated K+ currents in pulmonary artery smooth muscle cells: role of 5-HT2A receptors, caveolin-1, and KV1.5 channel internalization.</a> Circulation Research. 98 (7), 931-938 (2006).</li><li>Ganitkevich, V., Isenberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+potential+modulates+inositol+1,4,5-trisphosphate-mediated+Ca2++transients+in+guinea-pig+coronary+myocytes.">Membrane potential modulates inositol 1,4,5-trisphosphate-mediated Ca2+ transients in guinea-pig coronary myocytes.</a> Journal of Physiology. 470, 35-44 (1993).</li><li>Yamagishi, T., Yanagisawa, T., Taira, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K++channel+openers,+cromakalim+and+Ki4032,+inhibit+agonist-induced+Ca2++release+in+canine+coronary+artery.">K+ channel openers, cromakalim and Ki4032, inhibit agonist-induced Ca2+ release in canine coronary artery.</a> Naunyn-Schmiedeberg's Archives of Pharmacology. 346 (6), 691-700 (1992).</li><li>Okada, Y., Yanagisawa, T., Taira, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRL+38227+(levcromakalim)-induced+hyperpolarization+reduces+the+sensitivity+to+Ca2++of+contractile+elements+in+caninse+coronary+artery.">BRL 38227 (levcromakalim)-induced hyperpolarization reduces the sensitivity to Ca2+ of contractile elements in caninse coronary artery.</a> Naunyn-Schmiedeberg's Archives of Pharmacology. 347 (4), 438-444 (1993).</li><li>Xia, J., Little, T. L., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pathways+of+the+conducted+electrical+response+in+arterioles+of+hamster+cheek+pouch+in+vitro.">Cellular pathways of the conducted electrical response in arterioles of hamster cheek pouch in vitro.</a> American Journal of Physiology. 269 (6 Pt 2), H2031-H2038 (1995).</li><li>Jaggar, J. H., Porter, V. A., Lederer, W. J., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sparks+in+smooth+muscle.">Calcium sparks in smooth muscle.</a> American Journal of Physiology-Cell Physiology. 278 (2), C235-C256 (2000).</li></ol>

本文提供了如何使用锋利的微电极浸渍法从罐化容器制备中记录Vm的必要步骤。该方法被广泛使用,并提供高质量、一致的Vm记录,回答广泛的实验问题。
此处介绍了一些关键注意事项和故障排除步骤,以确保该方法成功。微电极的质量(其锐度和电阻)及其穿透的细胞过程影响Vm的稳定性和精度。如果信号持续漂移或高于或低于放大器的录制范围,则极有可?...

细胞的膜电位(Vm)是指等离子膜中离子电荷的相对差异以及膜对这些离子的相对渗透性。Vm由离子差分分布生成,并由离子通道和泵维护。Ion通道,如K+,Na+和Cl+对休息的Vm贡献很大。血管平滑肌细胞 (VSMC) 表示四种以上不同类型的 K+通道1,两种类型的电压门控 Ca2+通道 (VGCC)2,两种以上的 Cl+通道3,4,5、储存操作的 Ca2+通道 6、拉伸激活的阳离子通道7、8 和电原钠-钾 ATPase 泵9在其等离子膜中,所有这些都可能是参与Vm的管制。
VSMC 的 Vm取决于流明压力。在非加压容器中,Vm从 -50 到 -65 mV 不等,但在加压动脉段,Vm范围为 -37 到 -47 mV10。血管内压力升高导致VSMC去极化11,降低VGCC开口的阈值,并增加钙的流入,促进肌原性音调12的发展。相反,在被动或非加压容器中,由于高K+通道活性,膜超极化会阻止VGCC打开,导致钙进入受限和细胞内钙的减少,导致血管音13.因此,Vm由于流明压力的变化似乎在血管张力发展中起着至关重要的作用,而VGCC和K+通道在Vm的调节中起着至关重要的作用。
Vm因容器类型和物种而异。Vm是 -54 × 1.3 mV 在豚鼠优越的脑动脉条14,-45 × 1 mV 在大鼠中脑动脉在 60 mmHg 流明压力12,和 -35 × 1 mV 在大鼠腹腔动脉在 40 mmHg 流明压力15.未拉伸大鼠淋巴肌肉记录的休息 Vm是 -48 × 2 mV16。脑VSMC的Vm比外周动脉更负。相比之下,据报道,猫科脑动脉的Vm约为-70 mV,而据报道,脑动脉和冠状动脉分别有-49和-58 mV,分别为17、18。血管病床Vm的差异可能反映了电通道和电基钠钾泵的表达和功能的差异。
Vm中的增减分别称为膜去极化和超极化。Vm中的这些变化在许多生理过程中起着核心作用,包括电道门控、细胞信号、肌肉收缩和运动电位传播。在固定压力下,许多激活K+通道的内源和合成血管扩张剂化合物导致膜超极化,导致血管化1,13。相反,持续膜脱极极基在激动剂诱导或受体介导的血管收缩19中至关重要。Vm是一个关键变量,它不仅通过 VGCC13调节 Ca2+流入,还影响内部存储20、21和 Ca2+的 Ca2+的释放,合同装置22。
虽然有几种方法可以记录不同细胞类型的V m,但从罐体容器的微电极浸渍法收集的数据似乎比从分离的VSMC获得的数据更具生理性。当使用电流钳位方法从隔离的VSMC中记录时,Vm在VSMC24中被视为自发瞬态超极化。隔离的VSMC不在同步体中,串联电阻的变化可能导致Vm的振荡行为。另一方面,当从完整血管记录Vm时,振荡行为是不能观察到的,这可能是由于VSMC之间的细胞接触,这些细胞在动脉中是同步的,并且在整个血管中求和,导致稳定的Vm 24.因此,使用标准微电极浸渍技术从加压容器测量V m,与生理条件相对接近。
从可分离的血管记录Vm可以提供重要信息,因为同步的VSMC的Vm是血管音调和血流的主要决定因素之一,而Vm的调制可以提供一种拉升或收缩血管。因此,了解记录 Vm所涉及的方法至关重要。本文使用微电极浸渍法描述了从可熔化中脑动脉(MCA)的Vm细胞内记录。该协议将描述如何准备MCA,微电极,设置电表,并执行记录Vm的浸渍方法。此外,还讨论了代表性数据、使用此方法时遇到的常见问题和潜在问题。

<table>
	<tbody>
		<tr>
			<td>Dissection instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aneshetic Vaporiser</td>
			<td>Parkland scientific</td>
			<td>V3000PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon Instruments Inc., NY</td>
			<td>Eclipse Ti-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Kleine Guillotine Type 7575</td>
			<td>Harvard Apparatus, MA</td>
			<td>73-198</td>
			<td></td>
		</tr>
		<tr>
			<td>Littauer Bone Cutter</td>
			<td>Fine science tools</td>
			<td>16152-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria MC40 Ultra Fine Forceps</td>
			<td>Fine science tools</td>
			<td>11370-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors Sharp-Blunt</td>
			<td>Fine science tools</td>
			<td>14008-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture</td>
			<td>Harvard Apparatus</td>
			<td>72-3287</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine science tools</td>
			<td>15018-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophysiology Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Charge-coupled device camera</td>
			<td>Qimaging, , BC</td>
			<td>Retiga 2000R</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential electrometer amplifier</td>
			<td>WPI</td>
			<td>FD223A</td>
			<td></td>
		</tr>
		<tr>
			<td>In-line pressure transducer</td>
			<td>Harvard Apparatus, MA</td>
			<td>MA1 72-4496</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Thor labs</td>
			<td>PCS-5400</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrodes</td>
			<td>Warner Instruments LLC, CT</td>
			<td>G200-6,</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Fil (Microfiber syringe)</td>
			<td>WPI</td>
			<td>MF28G67-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode holder</td>
			<td>WPI</td>
			<td>MEH1SF</td>
			<td></td>
		</tr>
		<tr>
			<td>Myograph</td>
			<td>Living Systems Instrumentation, VT</td>
			<td>CH-1-SH</td>
			<td></td>
		</tr>
		<tr>
			<td>Puller</td>
			<td>Sutter Instrument, San Rafael, CA</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration-free table</td>
			<td>TMC</td>
			<td>3435-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Softwares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex 10</td>
			<td>Molecular devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p Clamp 10</td>
			<td>Molecular devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Nikon, NY</td>
			<td>NIS-elements</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P4504</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 </td>
			<td>Sigma</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H7006</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S0751</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
	</tbody>
</table>

microelectrode impalement method to record membrane potential from a cannulated middle cerebral artery

血管平滑肌细胞的膜电位 (Vm) 决定血管张力,从而决定血液流向器官。在疾病条件下调节Vm的电通道和电源泵的表达和功能的变化可能会改变V m、血管张力和血流。因此,对电生理学的基本理解和准确记录Vm在健康和疾病状态的必要方法是必不可少的。这种方法将允许使用不同的药理剂调制Vm以恢复V m。虽然有几种方法,各有其优缺点,本文提供了使用微电极浸渍法记录从罐化阻力血管(如中脑动脉)的Vm的协议。中脑动脉在造血室中获得造血性,血管壁使用高电阻微电极。Vm信号通过静电计进行收集、数字化和分析。该方法提供血管壁Vm的精确读数,不损坏细胞,也无需改变膜电阻。

该方法可以可靠地用于在罐式容器中记录Vm。图1A中介绍了一个描述如何从大脑中分离MCA的简要过程。将大脑与头骨分离后,MCA被解剖出来,并放置在含有低钙PSS的培养皿中。附加的部分结缔组织也使用弹簧剪刀和钳子与 MCA 一起解剖,以防止在隔离过程中对 MCA 造成损伤。仔细,结缔组织也被移除,解剖的MCA准备转移到近影。MCA 安装在导管上,在表?...

Watch this Scientific Journal Video about Microelectrode Impalement Method to Record Membrane Potential from a Cannulated Middle Cerebral Artery at JoVE.com

Microelectrode Impalement Method to Record Membrane Potential from a Cannulated Middle Cerebral Artery

本文的主要目的是提供如何使用微电极浸渍法记录中脑动脉膜电位(Vm)的详细信息。可加法的中脑动脉得到平衡,以获得造血性音,使用高电阻微电极刺穿血管壁。

微电极镀膜法记录从可吸入性中脑动脉的膜电位

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ...

该协议可用于测量正常和患病血管的膜电位，并评估调节膜电位、血管色调和血液流动的药理剂。这种技术产生的膜电位接近生理范围，因为血管平滑肌肉在血管中是同步的。在开始该步骤之前，将双通道差分电表放大器放在所需位置的容器室附近。使用 BNC-BNC 电缆将放大器通道 A 或 B 的输出连接到数字化器的通道输入。将探针安装在微操纵器中，将微操纵器转向无振动桌子上的显微镜和图像仪。如手册所述，将放大器前部的旋钮和开关放在实验的适当位置。然后用适当的电极将浴场连接到放大器的电路接地，并确保保持架接地到放大器的机箱上。要准备硅酸盐玻璃微电子，请使用标准拉拔器实现直径小于一微米的短、渐进、8 至 10 毫米锥度，循环两次，以实现更高的电阻和较小的尖端。使用超纤维注射器向微电杆填充三摩尔氯化钾，在注射过程中缓慢缩回柱塞，以便让液体充满空间，并防止微电子内形成气泡。将满载的微电极放在微电极支架上，小心而坚定地通过钻孔将电极柄推入支架。用实验室组织去除多余的液体，然后将电极支架组件连接到放大器探头。执行电极测试以测量电极电阻。然后打开录制软件，为文件分配名称，然后保存该文件以进行将来分析。接下来，用蒸馏水冲洗图像室几次，然后用五毫升的正常生理盐溶液（PSS）装入室。使用 5 或 10 毫升注射器，用过滤的正常 PSS 填充玻璃罐和附加的管子，而不引入任何气泡，并使用钝钳在两个 10-0 单丝尼龙缝合线中制作半结。然后，在解剖显微镜下使用解剖钳，将部分封闭的缝合结放在两个牙尖上稍远离尖端。对于中脑动脉隔离，使用弹簧剪刀和钳子来识别和解剖中脑动脉的未支段，内径为100至200微米，大鼠大脑。使用细钳将中脑动脉安装到玻璃罐上，并拧紧缝合线，将动脉固定到动脉上。关闭端管，使动脉内没有流量，并将流入移液器连接到 PSS 储液罐。使用安装在倒置显微镜和成像软件上的电荷耦合设备摄像机可视化可调中脑动脉。将 MCA 的轴向长度设置为近似长度，使其看起来既不硬，也不松弛。在 37 摄氏度下，用 95% 的氧气和 5% 的二氧化碳平衡沐浴液。将放大器的地面浸入刻画的 PSS 中。照亮容器室，并通过显微镜在沐浴液中可视化微选择尖端。使用微操纵器，将微电杆的尖端移近血管外壁，然后开始记录。缓慢地将微电子的尖端向容器移动，瞄准容器的中心。当尖端靠近容器时，以一个快速运动向前推进电极，刺穿肌肉的膜。一旦膜被穿透，膜电位的变化可能会观察到。膜被刺穿后，不要触摸微操纵器。记录膜电位变化后，使用操纵器在一次快速移动中移除微选择，并在保存数据文件之前停止记录。当快速偏转至负值时，当膜电位稳定至少 30 秒，并且当移除电极时，电压突然返回为零毫伏时，则计算是成功的。成功浸渍和膜电位稳定后，感兴趣的药物可以注入到浴池中，并记录膜电位的变化。在这个具有代表性的实验中，氯化钾灌注使膜脱极约6毫伏，而用钙依赖的活化剂大量导电钙活性钾通道使膜高极化近4毫伏。要记住的最重要的事情是拉高耐微电杆，具有短，逐渐锥度小于一微米直径。

Research

JoVE Journal

Medicine

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

建模在小鼠体内的行程 - 中东与灯丝模型大脑中动脉闭塞

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

科研

医学

Mouse Model of Middle Cerebral Artery Occlusion

大脑中动脉闭塞的小鼠模型

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

腔内中东大脑中动脉闭塞（MCAO）缺血性中风模型在小鼠体内的激光多普勒血流指导

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

在小鼠永久性大脑中动脉结扎术中的应用

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

内皮素-1诱导大脑中动脉闭塞模型缺血性中风激光多普勒血流仪在大鼠指导

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

利用颅窗口，在可视化的大脑中动脉内皮素-1诱导大脑中动脉闭塞

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

优化系统的脑灌注监测在中风模型大鼠的大脑中动脉闭塞腔内

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

造型中风小鼠：在远端大脑中动脉永久性混凝

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...