作者没有任何致谢。

这里描述的所有规程由奥地利政府道德委员会批准动物研究 (Bundesministerium Wissenschaft, Forschung 和 Wirtschaft, WF/伏/3b) 和符合协会为实验室评估动物使用动物保育指南。用于这种方法的老鼠是雌性, 12 周大的路易斯鼠。在整个协议中使用无菌仪器。
1. 材料制备
<ol><li>
导管的制造
	<ol>
<li>将导管 (聚乙烯导管 PE-50) 在适当长度 (20 到25厘米) 上, 以适合动物的大小。 注: 留一些额外的长度隧道和更容易处理。</li>
		<li>耀斑一个结束了一个打火机得到一个圆润的提示。检查导管的最后合适的开口和钝端。</li>
		<li>将一个2毫米长的硅胶管放在导管上方, 直到它位于喇叭的正下方。</li>
	</ol>
</li>
	<li>
电极的制造
	<ol>
<li>准备一个20-25 厘米的聚四氟乙烯涂层钢丝 (长短取决于动物的大小)。</li>
		<li>准备一个银线长度2厘米和扭曲的两端, 直到一个小循环保持。在一端剥去2毫米的聚四氟乙烯绝缘。焊接扭曲的银线结束的剥离钢丝。</li>
		<li>在涂布区应用常规指甲油。在涂敷区域上准备4毫米长的聚乙烯导管, 并通过将热量应用于压缩钳来密封末端。</li>
	</ol>
</li>
</ol>2. 动物制剂
<ol><li>
麻醉
	<ol>
<li>使用适当的麻醉鸡尾酒机构批准的全身麻醉。</li>
		<li>准备麻醉鸡尾酒与 medetomidine (0.15 毫克/千克), 咪唑安定 (0.08 毫克/千克), 和芬太尼 (0.01 毫克/千克), 并注射注1毫升注射器与27测量针。</li>
	</ol>
</li>
	<li>
手术准备
	<ol>
<li>用电动剃须刀刮伤腹部, 包括肩胛骨的生殖器区域和背部区域。</li>
		<li>先用70% 乙醇消毒腹部和颈部区域, 然后用聚维酮碘溶液3交替擦洗。</li>
	</ol>
</li>
</ol>3. 膀胱导管植入
<ol><li>在第三和第四奶嘴 (大约2到2.5 厘米长) 的水平上进行低线剖腹手术, 使用手术刀为皮肤和外科剪刀为腹部肌肉。</li>
	<li>通过在颅尾的方向引导腹壁并将其固定在膀胱的后面, 以避免重新定位, 从而使膀胱显露出来。</li>
	<li>使用6-0 不可吸收的单丝缝线和锥形针尖针在膀胱圆顶周围放置一个包线缝合。</li>
	<li>切开膀胱圆顶在钱包串内, 或者由手术刀尖或18G 针, 插入膀胱导管 (参见协议步骤 1)。</li>
	<li>插入导管预与无菌生理0.9% 氯化钠溶液, 并仔细收回膀胱导管, 直到扩口的导管是位于膀胱圆顶以下。</li>
	<li>在导管周围固定包线缝合, 并在导管体周围进行停止缝合, 以进行进一步的固位。</li>
	<li>通过导管缓慢填充膀胱, 用无菌生理0.9% 氯化钠溶液, 检查膀胱穹窿的渗漏。</li>
</ol>4. 尿道括约肌电极植入术
<ol><li>准备三电极植入 (见协议步骤 1)。</li>
	<li>标记一电极用色的永久毛毡笔为未来空电极的进一步证明。用70% 乙醇消毒电极。 注: 导管不适合于热化学灭菌程序。相反, 冷杀菌的电极24小时。</li>
	<li>用外科剪刀将腹部切口延伸至耻骨, 但不要切开耻骨联合。</li>
	<li>通过尿道两侧的细钳, 识别尿道并创建钝袋, 但要避免血管或神经的损伤。</li>
	<li>确定适当的脂肪囊靠近尿道在这个窗口。</li>
	<li>使用6-0 不可吸收的单丝缝合线, 通过双边方式将电极固定在适当的脂肪袋上。 注意: 电极的最后位置应该是双侧的在尿道的中部区域。</li>
	<li>将两个电极与6-0 不可吸收的单丝缝合在一起。</li>
	<li>将标记的空电极缝合到尿道的距离内的腹壁肌肉。将所有三电极与一条缝合线绑在一起。</li>
</ol>5. 隧道
<ol><li>在肩胛骨之间做一个小的皮肤切口为隧道。</li>
	<li>隧道电极线到颈部, 并做最后检查正确放置, 并验证在隧道后电极的位置。</li>
	<li>隧道膀胱导管到肩胛骨水平-注意膀胱, 而隧道导管, 以避免膀胱扭曲。为了达到这个目的, 在进入膀胱穹窿前按住导管, 以避免膀胱扭曲。</li>
	<li>用 4-0 polyfilament 可吸收缝合线连续或间断缝合, 关闭腹部肌肉。通过中断的床垫缝合关闭皮肤切口。</li>
	<li>将动物伸展至其全长, 使电极线和导管的最大膨胀。</li>
	<li>将导管和电极丝固定在肩部肌肉的缝合线上。</li>
	<li>用4-0 缝线缝合皮肤。</li>
</ol>6. 吊带配件
<ol><li>在动物头上拉上安全带, 将前肢拉到两个硅胶条之间的末端位置, 以适应动物。在与分销商进行手术之前, 检查一下动物的重量。</li>
	<li>隧道的膀胱导管的中心孔的线束和电极通过定制孔的钻头调整, 以电极线的大小。</li>
	<li>通过拉动硅胶条来调节安全带。调整安全带, 使其不太松散, 但确保一些空间仍然保持移动能力的大鼠。</li>
	<li>使用电缆带固定硅条。</li>
	<li>将膀胱导管的长度削减到3厘米以上的线束, 连接到23克塞子, 最后修复它的安全带。</li>
</ol>7. 电极连接器的制造
<ol><li>准备三小的热收缩管 (一个不同的颜色为零电极)。准备两个进一步的热收缩管在一个合适的较大的大小。</li>
	<li>缩短电极导线的长度到最佳长度, 以便能够在以后插入女性插头, 而不太松或太短。剥去三根电线 (约2毫米) 的聚四氟乙烯绝缘, 并将钢丝绳捻成一个字符串。</li>
	<li>将大型热收缩管置于所有电线上, 将小管单独放置在所有三根电线上, 并使用有色管作为空电极。</li>
	<li>焊锡电极到男性3连接插头。将空电极放在中间, 将三小的单个管收缩到焊接区域之上。</li>
	<li>收缩的第一个较大的管在年底的个别管尾和最后大管到男性插头边界。</li>
	<li>将雄性插头连接到固定在吊带上的母接头上, 并用一条胶带固定以进一步安全。</li>
</ol>8. 术后护理
<ol><li>清洁手术区, 用聚维酮碘消毒。</li>
	<li>将动物放在加热垫上直到清醒, 然后根据当地动物护理指南, 在手术期间和术后给0.9% 氯化钠溶液进行水替代。</li>
	<li>管理镇痛药 (昔1毫克/千克) 和抗生素 (sulfadoxinum 200 毫克, trimethoprimum 40 毫克, 15 毫克/千克) 作为联合注射溶液皮下注射。每天两次, 给镇痛药 (早晚), 每天一次给予抗生素, 随后五天。</li>
	<li>继续使用相同剂量的抗生素, 在整个随访期间, 每周注射两到三次。</li>
	<li>每天检查适当的吊带, 并进行外科实地检查, 特别是颈部区域。调整线束, 如果它变得太紧, 仔细地拉在硅胶条。</li>
	<li>每周定期冲洗导管, 避免阻塞。</li>
</ol>9. 压测量的准备
<ol><li>执行第一次压测量后六后手术天。 注: 早期的测量可以受镇痛药和/或尿囊刺激, 由于导管植入。</li>
	<li>打开主开关、计算机和肌电信号放大器。</li>
	<li>用室温加热0.9% 氯化钠填充注射器泵。以下行方式依次打开三路连接器, 从泵开始, 然后冲洗管子。 注: 仔细检查剩余气泡, 因为气泡会改变压的测量。</li>
</ol>10. 校准
<ol><li>启动程序 uroflowmeter 软件, 然后转到校准。</li>
	<li>关闭泵和动物的三路连接器。</li>
	<li>打开阀门到连接的压力表, 并按零在程序中。</li>
	<li>调整压力在压表到100柱, 并按100柱按钮的程序。按 "确认" 以保存校准调整。窗口将自动关闭。</li>
	<li>关闭阀门的压力表和打开三路连接器从泵到动物准备好测量。</li>
</ol>11. 动物数据库 (动物 DB)
<ol><li>要注册动物, 给动物一个简明的 ID。</li>
	<li>输入进一步的数据, 如 SCI 的日期, 开始治疗, 导管植入日期, 实验组, 和动物的出生年月。</li>
	<li>要最终记录数据, 请按 "存储记录" 并保存到文件。</li>
</ol>12. 记录前的测量设置
<ol><li>按 "开始" 按钮启动软件程序。</li>
	<li>选择动物和压重量秤和零压力。 注意: 在将动物放入抑制剂时要注意, 这样就不会有电缆卡住, 因为这可能会导致肌电信号线的移除。</li>
</ol>13. 动物制剂
<ol><li>拔下导管插头, 从吊带上拔下肌电信号插头。</li>
	<li>把老鼠放在抑制剂。关闭抑制剂, 用钳锁安全带。</li>
	<li>把导管和肌电信号插入抑制剂。</li>
	<li>将抑制剂放置在猫单元中。把尾巴穿过管子, 用胶带固定管子, 避免运动。</li>
	<li>将男性肌电信号插头连接到记录女性肌电信号连接器。</li>
	<li>再按零压力, 然后将导管连接到灌装/录音管。检查软件中的压力。 注: 压力应该是正数, 大约5-10 厘米 H2O 在基线。如果压力是负的, 断开导管从套管, 按零压力, 然后重新连接导管。</li>
</ol>14. 记录
<ol><li>如果导管和肌电信号连接, 按下运行泵和录音。</li>
	<li>使灌装速度适应μ l/分钟的实验需要。</li>
	<li>记下记录开始的时间和日志簿中的室温。</li>
	<li>要停止压记录, 请按下运行泵并再次按下记录停止泵。 注: 水泵将关闭, 泵的绿灯熄灭。</li>
	<li>断开导管并使用23G 塞子插头关闭导管末端。断开肌电信号电缆。</li>
	<li>打开抑制剂的前面, 引导老鼠走出抑制剂。 注意: 小心地处理老鼠, 并监视电线, 以避免电缆的任何锁定。</li>
	<li>将导管插回安全带。将肌电插头重新插到吊带上, 并在插头周围放置一条胶带。 注: 如果动物有病理性下尿路状况, 请在末端手动表达膀胱以避免膀胱 overdistention。</li>
	<li>把动物放回笼子里</li>
	<li>按退出键关闭程序。</li>
	<li>清洁抑制剂和烧杯。</li>
	<li>关闭泵和动物出口的三路连接器。</li>
	<li>通过主交换机关闭计算机、肌电信号单元和系统。 注意: 数据保存在单独的文件夹 "记录的数据" 与被测量的鼠 ID 的子文件夹文件。单个记录按日期在单个 rat ID 文件夹中排序。</li>
</ol>

<ol>
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作者没有什么可透露的。

本协议描述了永久性导管和尿道括约肌电极植入的手术过程和压记录技术在清醒, 轻度抑制大鼠包括同时分析膀胱和尿道外括约肌。
手术中的关键步骤是仔细植入膀胱导管, 避免渗漏和广泛操作。此外, 对尿道外括约肌的双侧电极进行精确的植入, 对于外括约肌的声音测量至关重要。在植入后对手术场进行严密检查对维持动物的健康也是必不可少的。后续的抗生素覆盖有助于预防沿导管线感染, 以及发生尿路感染。
在压测量期间, 处理过的老鼠会比以前没有处理过的老鼠更镇定、更放松。因此, 压记录的结果可能会有所不同。此外, 抑制剂为鼠提供了一个封闭的空间与一个黑暗的前沿区域感到更舒适, 从而减少压力水平。在其他被出版的醒的压测量, 老鼠在测量的笼子可以自由地移动。然而, 这在测量过程中具有较高的人工风险, 可能会增加记录的时间, 同时也会提高动物的应力水平。在健康的大鼠, 最佳的压测量可以复制在多个测量时间点在后续。在压测量过程中, 通常出现的问题是避免导管, 或者是协议的步进式传导中的一个错误。如果发生技术或软件错误, 则强烈建议重新初始化压测量和协议的逐步重复, 以便进行故障排除。
这项技术的局限性是压记录的动物间变异性, 植入导管引起的膀胱组织结构改变, 这可能会阻碍组织的组织学或分子生物学检查,和动物在随访期间的单一住房。此外, 这项技术只在雌性大鼠中进行了试验, 对雄性大鼠的适用性和结果尚未进行检查。
这项技术的主要优点是同时测量膀胱和外尿道括约肌, 以及清醒的测量设置。因此, 对清醒动物下尿路的一个更具平移性的检查是可用的, 相比之下, 在麻醉的单一, 终端压分析5,6,9,10。此外, 随着这种方法, 一个较低的泌尿道功能障碍或病理的进展可以遵循在同一动物随着时间的推移, 以及治疗的成功。特别是, NLUTD 可以被审查在一个时间路线, 是不可能的到一个充分的程度与共同的压技术2。
最后, 提出的手术和压测量用于多, 人工减少下尿路的分析, 包括膀胱和外尿道括约肌在清醒大鼠的相互作用。

为了分析尿液的储存和排尿功能和功能障碍, 大多数研究都使用了啮齿动物模型。通过连续的反应激活, 排尿产生。这些反射的协调对于有效地排尿1至关重要。压记录技术为分析其神经控制下的膀胱功能提供了有价值的工具1。
大多数传统的 cystometries 在大鼠是作为一个单一的, 最终分析麻醉, 主要是聚氨酯2, 并专注于膀胱完全。然而, 在某些病症中如神经源性下尿路功能障碍 (NLUTD), 不仅膀胱, 而且排尿口, 外部尿道括约肌, 是功能失调的3,4。这使 NLUTD 困难跟随-, 如果仅膀胱在一个单一压测量被审查。为了获得与人类相媲美的可靠结果, 必须准确测量膀胱和尿道外括约肌功能及其相互作用2。此外, 对清醒大鼠进行功能分析非常重要, 因为麻醉很可能会改变膀胱功能2,5,6。一个好的压记录在醒的动物是依据为识别膀胱作用和故障7。
使用的小动物 cystometry 驻地 (例如, 猫 cystometry 驻地 (CCS)) 是一个单位执行压分析在小醒的动物8。通过永久性膀胱导管和植入的外部尿道括约肌电极, 可在较长时间内进行重复性测量2。因此, CCS 为啮齿动物模型中的非神经源和 NLUTD 评价提供了一个有价值的工具, 在这种模式中, pathomechanisms 可以在短期或中期随访中改变。此外, 该方法包括通过使用抑制剂在清醒大鼠中进行膀胱测量来减少压分析。
在本文中, 我们描述了永久性植入膀胱导管和外尿道括约肌电极的手术方法, 以及在清醒大鼠的压测量。

<table>
	<tbody>
		<tr>
			<td>Polyethylene tubing PE 50</td>
			<td>Becton Dickinson</td>
			<td>427411</td>
			<td>Catheter</td>
		</tr>
		<tr>
			<td>Prolene 6-0 (BV-1, 9.3 mm, 3/8c)</td>
			<td>Ethicon</td>
			<td>EH7403H</td>
			<td>Suture</td>
		</tr>
		<tr>
			<td>Teflon coated steel wire</td>
			<td>Cooner wire</td>
			<td>AS631</td>
			<td>Electrode material</td>
		</tr>
		<tr>
			<td>Silver wire 0.250 mm</td>
			<td>World Precision Instruments</td>
			<td>AGW1030</td>
			<td>Electrode material</td>
		</tr>
		<tr>
			<td>Rotilabo - PVC tube</td>
			<td>Carl Roth</td>
			<td>97241</td>
			<td>Harness</td>
		</tr>
		<tr>
			<td>Vicryl rapide 4-0 (P-3, 13 mm, 3/8c)</td>
			<td>Ethicon</td>
			<td>V4940H</td>
			<td>Suture</td>
		</tr>
		<tr>
			<td>Quick Connect Single Harness</td>
			<td>SAI Infusion Technologies</td>
			<td>QCH-23CW</td>
			<td>Harness</td>
		</tr>
		<tr>
			<td>Shrinking tubes</td>
			<td>ChiliTec</td>
			<td>17894</td>
			<td>Electrode soldering</td>
		</tr>
		<tr>
			<td>Soldering wire Pb60 Sn40</td>
			<td>Stannol</td>
			<td>LD0029</td>
			<td>Electrode soldering</td>
		</tr>
		<tr>
			<td>Fluxing agent 157</td>
			<td>Castolin Eutectin</td>
			<td>157 0150</td>
			<td>Electrode soldering</td>
		</tr>
		<tr>
			<td>Conn Unshrouded Header HDR 3 POS, 2.54mm Solder ST Thru-Hole Box</td>
			<td>Preci-dip</td>
			<td>801-87-050-10-001101</td>
			<td>Electrode soldering</td>
		</tr>
		<tr>
			<td>Conn Socket Strip SKT 50 POS 2.54mm, Solder ST Thru-Hole Box</td>
			<td>Preci-dip</td>
			<td>890-18-003-10-001101</td>
			<td>Electrode soldering</td>
		</tr>
		<tr>
			<td>Rat Cystometry Package (contains pump, scale, pressure transducer, hardware for cystometric analysis)</td>
			<td>Catamount Research and Development Inc.</td>
			<td>CAT-CYT-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential amplifier with active headstage</td>
			<td>AD instruments</td>
			<td>DP-311</td>
			<td>EMG amplifier</td>
		</tr>
		<tr>
			<td>Restrainer Medium size for rats 200-300 g</td>
			<td>emka Technologies</td>
			<td>HLD-RM</td>
			<td></td>
		</tr>
		<tr>
			<td>Uro Dyn Software</td>
			<td>Z&#252;rich of University</td>
			<td></td>
			<td>MTA-based</td>
		</tr>
		<tr>
			<td>Female rats (Strain Lewis) 12 weeks of age</td>
			<td>Charles River, Sulzfeld, Germany</td>
			<td></td>
			<td>animals</td>
		</tr>
	</tbody>
</table>

cystometric and external urethral sphincter measurements in awake rats with implanted catheter and electrodes allowing for repeated measurements

下尿路功能主要通过对啮齿动物的压膀胱功能分析来评估。常规 cystometries 通常在聚氨酯麻醉下进行终端分析。众所周知, 麻醉药能影响膀胱功能。因此, 该技术的目的是进行压测量的膀胱和外尿道括约肌轻度克制清醒大鼠。为此, 膀胱导管被植入膀胱圆顶。随后, 两个电极被植入双侧的外部尿道括约肌和一个接地电极缝合的非反应性骨骼肌。膀胱导管和三电极最终通过隧道皮下注射到颈部区域, 并贴在安全带上。采用这种技术, 可以在同一动物的多个时间点测量下尿路, 以评估下尿路功能。该技术的主要应用是在清醒健康的大鼠和诱导的疾病或损伤后同时膀胱和外括约肌功能的随访。此外, 随后的下尿路监测可以进行评估的疾病/伤害和监测治疗效果。

在图 1中显示了唤醒压测量过程的示意图, 并在图 2中显示了膀胱导管植入的内部解剖。手术需要2小时, 术后镇痛和抗生素, 在手术后五天内包括疼痛和感染。此后没有发现任何疼痛的迹象。每天两次仔细检查腹部、腹部缝合和颈部缝合是维持动物健康所必需的。线束控制 (位置和松紧度) 应在头五天和以后定期进行一次。腹部缝合线可以删除在 10th后手术天。
我们在手术后的前10天使用柔软的非木本寝具来避免炎症。床上用品每周至少更换两次, 以进一步降低炎症的风险。动物被保存在单一的住房中, 因为群体住房增加了利用、导管或电极电缆咬住笼友的风险。
导管应至少每周冲洗一次, 无论是在压测量过程中, 还是用1-3 毫升无菌0.9% 氯化钠以低输液速度手动冲洗导管 (图 3)。定期的抗生素覆盖的动物进一步减少感染的风险和泌尿系结石的形成。监测体液摄取是防止泌尿系结石形成的又一重要点。柠檬酸在低浓度 (2-3%) 是通过导管或进入饮用水 intravesically, 以防止结石形成。
手术的成功率, 以及维持膀胱导管和电极完整, 大约80%。在余下的20% 的情况下, 主要的问题是从插头的电极线脱离。因此, 小心地将电极线连接到线束上是避免电极损耗的关键。
压测量通常做, 直到三连续的排尿周期记录每测量, 采取在20到 40 min 之间, 取决于焦虑和处理状态的鼠。第一次压测量通常是在导管植入手术一周后进行。
压记录的主要读出参数是基线压力、阈值压力、最大逼尿肌压力、无效容积、平均流量、排尿时间、平均压力、膀胱顺应性以及同时读出外部尿道括约肌肌电-活动 (图 4)。
连续的压测量在后续的期间可以执行至少四星期在手术以后。如果导管线经常冲洗, 导管堵塞是没有问题的。在整个随访期间, 应进行大鼠的常规处理和光控。
如果导管是避免或阻断, 膀胱灌注压力将增加线性到非常高的压力 (100 厘米 H2O)。在这种情况下, 应停止灌装和可见的导管端应检查康祺。如果未发现康祺, 则应检查导管是否有堵塞的插座。为此, 导管可以手动冲洗通过导管。如果液体不是容易地流动入膀胱, 拉扯和向前轻轻地可以被尝试。最后一次尝试, 酸性冲洗液 (柠檬酸 2-3%) 可用于尝试清除导管内的受阻区域。这个解决方案可能有更高的机会解除封锁, 然而, 膀胱将被激怒后, 成功冲洗和连续的测量应只执行两天后冲洗与酸性溶液。如果没有液体能被冲洗入膀胱, 导管永久地被阻拦, 并且没有进一步测量是可能的, 并且动物丢失为后续。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56506/56506fig1.jpg"> 图 1: 清醒大鼠压测量的原理图.此图已从2进行了改编。(a) 尿动力学设置的图示。(b) 实验室站进行尿动力学检查。(c) 外侧尿道括约肌肌电电极植入尿道、术中观察。(d) 膀胱导管植入时的膀胱穹观, 术中观察。(e) 在植入电极和导管后, 老鼠将配备安全带, 以安全地储存插头和连接器。(f) 人类尿。b-e中的数字与a中的图例相关。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56506/56506fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56506/56506fig2.jpg"> 图 2: 大鼠膀胱导管植入的内部解剖.此图已从8中修改。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56506/56506fig3.jpg"> 图 3: 大鼠导管线的冲洗.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56506/56506fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56506/56506fig4.jpg"> 图 4: 导管植入后12天的动物尿动力学脉象.(a) 代表性的尿动力学追踪从一个天真的老鼠。顶部显示膀胱压力示踪, 在中间分泌的尿量追踪, 并在底部的外部尿道括约肌肌电追踪。(b) 缩放窗口从六十年代的一个天真的动物, 取自 (a)。一个重要的说法是, 排尿时的外部尿道括约肌肌电活动少于排尿前后。顶部显示膀胱压力示踪, 在中间分泌的尿量追踪, 并在底部的外部尿道括约肌肌电追踪。在底部, 一个热图显示与时间匹配的频率频谱图 (对应于当前时间点的频率)。红色代表高功率, 蓝色代表低功耗。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56506/56506fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements at JoVE.com

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

本协议首先介绍了膀胱导管与外尿道括约肌电极联合永久性植入的手术方法, 其次, 对膀胱及外尿道功能的测量在植入的清醒动物的括约肌。

压和外尿道括约肌测量在清醒大鼠植入导管和电极允许重复测量

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

cystometric-external-urethral-sphincter-measurements-awake-rats-with

Nanyang Technological University

Research

JoVE Journal

Biology

15.8K 次观看.  Paracelsus Medical University. 本协议首先介绍了膀胱导管与外尿道括约肌电极联合永久性植入的手术方法, 其次, 对膀胱及外尿道功能的测量在植入的清醒动物的括约肌。

视频: Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

膜片吸液管和夏普电极与一个可编程的拖轮

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

科研

生物学

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

赖氨酸去甲基化酶活性的定量测量中的应用的MassSQUIRM

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

CA同时测量人的心房肌细胞分离<sup&gt; +</sup&gt;瞬变和膜电流

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

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 ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

发展与表征<em&gt;体外</em&gt;微血管网络和内皮的定量测量的[Ca<sup&gt; 2+</sup&gt;]<sub&gt;我</sub&gt;和一氧化氮含量

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

新鲜分离和内联小鼠脑内皮细胞内钙和膜电位的同时测量

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Elegans生理应激反应的测量

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

用于硝酸盐和亚硝酸盐测量的大鼠骨骼肌匀浆的制备

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...

压和外尿道括约肌测量在清醒大鼠植入导管和电极允许重复测量

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