Name: novel approach for simultaneous recording of renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious, unrestrained mice.
Uploaded: 2018-02-14T12:30:00.000+00:00
Duration: 11 min 8 s
Description: Watch this Scientific Journal Video about Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice. at

S.M.H. 得到了加拿大卫生研究所 (卫生研究院)、加拿大心脏 & 中风基金会 (HSFC) 和艾伯塔省创新保健解决方案 (AiHS) 博士后研究金的支持;J.E.H. 得到国家心脏、肺和血液研究所 PO1HL-51971 的资助。

所有的实验程序都符合国家卫生研究院关于实验室动物的护理和使用指南, 并得到密西西比大学医学中心机构动物护理和使用委员会的批准。
1. 动物和住房
<ol><li>房屋老鼠 (24-35 克) 抵达后, 机构实验室动物设施。</li>
	<li>在温度和湿度控制环境下, 在实验协议的各个阶段提供老鼠标准啮齿动物和自来水广告随意。</li>
</ol>2. 植入式北美放射学会电极的定制制作
注: 在预定的手术过程中至少提前几天构造植入北美放射学会电极, 以适应固化和灭菌时间 (如下所述)。
<ol><li>切割三等长度的绝缘不锈钢多绞线, 每250毫米 (线直径0.0254 毫米裸, 0.14 毫米涂层)。使用手术刀刀片 (最好是 #11), 剥去大约15毫米的绝缘材料, 从每条长度的导线的一端露出底层的金属。<ol>
<li>焊锡一个单一的男性引脚连接器 (黄铜与镀金) 到裸露的两端只有两条电线, 以创建两极电极引线 (图 1A)。离开第三长度的电线裸露的结束。这将起到地线的作用。</li>
		<li>滑动短 (~ 2.0-2.5 厘米) 件1.6 毫米直径的热收缩油管在针连接器和导线, 完全覆盖新焊接接头之间的电线和针连接器。 注意: 将插入放大器 headstage 的 pin 连接器的尖端必须保持暴露。</li>
		<li>用一双小钳子或止血剂将电线放在热枪上方, 以收缩热敏油管, 并电绝缘针接头与导线之间的连接。对第二个导线/针脚连接器重复。</li>
	</ol>
</li>
	<li>切割200毫米聚乙烯油管长度 (PE 90; 内径0.86 毫米, 外径1.27 毫米)。将三根导线 (两条引线 + 地线) 组合在一起, 将未接触的端部引入 PE 90 油管, 将它们连接到油管的开口端 (图 1B)。 注: PE 90 油管起到保护电极导线和地线的作用。<ol>
<li>识别地线, 并通过 PE 90 鞘进一步拉它, 以区分它从两极电极引线。</li>
	</ol>
</li>
</ol>3. 电极尖端的构造
<ol><li>用解剖显微镜可视化电极线的未接触端。通过5毫米长的小聚乙烯油管 (PE 10, 内径0.28 毫米, 外径0.61 毫米) 将电极线的三松散端连接在一起。<ol>
<li>螺纹1.5 毫米这 PE 10 管材到三根电极导线。提前这油管休息2.0 毫米远离最初的 5 mm 的 PE 10。</li>
		<li>螺纹一秒钟1.5 毫米 PE 10 油管到两极电极的尖端, 导致覆盖和隔离的提示, 并与地线分开 (图 1C)。</li>
	</ol>
</li>
	<li>用剪刀修剪电线的多余长度。</li>
	<li>将 PE 10 油管的各个部分粘附在电极线上, 用一小滴液体配方氰胶水。在胶管的末端放置一个钝25口径的针, 以改善控制和减少溢出。<ol>
<li>将针尖放在 pe 10 和导线之间的连接处, 然后将一小滴胶水和可视化胶涂在 pe 油管内。</li>
		<li>允许胶水在一夜之间完全治愈。</li>
	</ol>
</li>
</ol>4. 精密制备记录电极尖端
<ol><li>用 #11 手术刀刀片从两极电极尖端和地线尖端剥离绝缘涂层。不要干扰或损坏底层的多股绞线, 因为这会影响北美放射学会信号的质量。</li>
	<li>用弯曲钳夹住5.0 毫米和1.5 毫米 PE 10 锚之间的构造电极, 弯曲导线形成90°角度 (图 1D)。 注: 此机动应定位双极电极引线以上的地线, 在最佳位置, 以摇篮神经丛。</li>
</ol>5. 锚固基座的施工
<ol><li>通过切割3厘米的聚乙烯油管 (内径2.70 毫米, 外径4.00 毫米), 构造一个稳定电极的基座, 使其在 exteriorization 的中肩胛骨区域。<ol>
<li>用镊子抓住油管, 然后用热枪将一端熔化。按垂直于冷金属表面的油管加热端, 创建圆脊或 "法兰"。</li>
		<li>将这个基座螺纹在所构造的电极上, 这样法兰就指向电极尖端。 注: PE 90 鞘和底座的组合将保护电极引线一旦形象化从动物。</li>
	</ol>
</li>
</ol>6. 已完成植入电极的灭菌
<ol><li>在植入前将已完成的电极单独包装在灭菌袋和臭氧杀菌 (草3) 中。 注: 与当地医院消毒设施有关的特定类型的灭菌袋和程序, 因为这不同的机构之间。</li>
</ol>7. 麻醉和手术准备
<ol><li>在手术开始前20分钟实施镇痛 (2 毫克/千克 meloxicam, 南卡罗来纳州)。将鼠标放置在注入100% 医学级氧气的感应腔内。调整蒸发器设置, 以增加异氟醚麻醉的百分比为 0.5, 达到4%。评估手术平面评估的反射反应, 温和的压力适用于脚趾或脚垫的前腿和后腿, 以及呼吸速度减慢。<ol>
<li>将动物转移到手术台上, 用1.5 至2% 异氟醚通过 nosecone 维持麻醉, 一旦到达手术平面, 不再显示脚趾捏反射。在整个手术过程中定期重复脚趾夹的反应, 评估呼吸率。将眼膏涂抹于眼部, 防止干燥。</li>
		<li>保持动物的正常体温在任何时候用凝胶填充的等温热垫和相应的手术表。在37°c 水浴中贮存等温垫, 并在手术期间根据需要更换护垫, 以保持生理体温。</li>
		<li>管理甘罗溴铵 (50-70 µg/千克, 皮下 (南卡罗来纳州)), 以防止在诱导麻醉后立即过度生产气道分泌物。在手术过程的中点 (步骤 9.1) 第二次管理这一剂量的甘罗溴铵。</li>
		<li>在无菌条件下进行所有外科手术。确保所有手术工具在预定手术前都已被蒸压。清洁手术领域如下所述 (7.2.1) 和保持不育整个过程。<ol>
<li>戴口罩、蒸压隔离袍和无菌、单用手套。清洁所有大型设备, 如鹅颈灯, 解剖范围和手术表与70% 乙醇。定期在手术过程中, 将70% 乙醇应用于外科手套, 以保证不孕。</li>
		</ol>
</li>
	</ol>
</li>
	<li>从动物的左侧面、腹颈区和背 midscapular 区去除毛发, 然后用小动物发脱毛膏 (敏感皮肤配方)。<ol>
<li>清洁这两个手术领域的皮肤与3交替应用的手术清洗液 (10% 聚维酮碘) 和70% 乙醇。准备手术现场, 最后应用手术清洗液。</li>
	</ol>
</li>
</ol>8. 北美放射学会电极的外科植入术
<ol><li>将鼠标放在右侧, 延髓末端指向外科医生的左侧, 露出动物的左侧面。用手术刀 (#11) 在 midscapular 区域的皮肤上做一个5毫米切口。 注: 这是北美放射学会电极引线将被形象化的站点。<ol>
<li>在左侧面的皮肤上做第二切口 (< 20 毫米), 垂直于脊柱和2毫米尾鳍到胸腔。隧道一个13G 不锈钢针皮下从这个切口到切口在背部出口部位。 注: 文件锋利的边缘, 使针离开光滑, 不切削边缘。</li>
		<li>通过13G 针通过灭菌的植入北美放射学会电极 (步骤 2-6)。将13G 针向后拉, 使电极尖端位于左侧腹部肌肉上。将电极的一段引线放在皮肤下, 并留下从背切口出现的剩余长度。</li>
	</ol>
</li>
	<li>将电极尖端置于侧面。在8.1.1 的皮肤切口上直接下腹部肌肉切开。用小的棉尖喷头将脂肪和结缔组织沿背部肌肉分开, 露出左肾。<ol>
<li>用微拉钩打开手术场, 并收回肾脏。不要伸展肾脏神经血管束, 这将不可逆转地损害肾脏神经和排除记录一个可行的北美放射学会信号。 注: 钢微拉钩可以从标准的回形针和长度4-0 丝绸。确保这些拉钩也与手术器械消毒, 以保持无菌技术。</li>
	</ol>
</li>
	<li>用高功率解剖显微镜可视化肾神经血管束。确定肾神经束, 通常 (但不总是) 沿肾动脉和静脉。用细直钳解剖周围组织的神经丛。 注: 肾神经束看起来不透明, 与 "绳子状" 反光的外观相比, 其独特性与淋巴管比较, 显得清晰。<ol>
<li>尽可能少的操纵神经丛。不要接触, 伸展或拿起神经束在任何时候。不要扰乱提供神经或肾淋巴管的细血管, 因为这会损害神经的生存能力, 并导致神经/电极周围持续的淋巴液聚集, 这会阻碍或彻底消除神经信号。</li>
		<li>将肾神经束保持完好, 这将有助于保持神经的长期生存能力, 并维持神经与电极之间的稳定接触 (即一段神经可能会随时间和自然身体运动而脱落电极)。</li>
	</ol>
</li>
	<li>将北美放射学会电极尖端引入腹部。调整其位置, 使两极电极尖端和地线垂直于肾神经血管束。进一步调整电极位置, 使地线与底层组织有良好的接触, 电极不压缩肾血管, 损害肾脏循环 (图 1D)。</li>
	<li>用角钳抬起肾神经束。在神经下方滑动电极尖端, 使神经与两根导线直接接触。<ol>
<li>在神经/双极导线和第三 (地线) 导线之间滑动一小片石蜡膜 (图 1D)。 注: 浸泡70% 乙醇消毒石蜡膜24小时, 在植入前用无菌生理盐水冲洗。</li>
		<li>用小的吸收矛除去神经/电极周围的任何血液或液体, 因为在神经或电极线周围留下的任何液体都会阻碍或熄灭神经信号。</li>
		<li>如果需要, 快速测试北美放射学会信号的质量 (下面描述的设置)。 注意: 这必须迅速完成, 因为暴露在空气中会使神经干燥并危及其生存能力。</li>
		<li>将双组分硅胶弹性体应用于神经/电极单元, 确保硅胶池在神经的下方和周围提供完全的电隔离 (即不只是在神经的顶端下降)。 注: 确保电极贴士也涂在硅胶上。地线应保持与底层组织的接触, 因此弹性体不需要在这根电线下面的池。避免使用不必要的大量的硅胶弹性体, 因为这可能会阻碍肾脏血流, 或成为脱落与自然身体运动与时间。</li>
		<li>让硅胶弹性体完全固化1-2 分钟, 然后用镊子小心翼翼地将硅胶的外边缘抬起, 并应用少量的液体配方手术粘合剂。 注: 注意不要使用过量的这种胶水, 因为它可能会损害循环或扩散到神经和损害其生存能力。</li>
	</ol>
</li>
	<li>用不连续的可吸收缝线闭合腹部切口 (5-0)。用同样的缝合材料以类似的方式关闭上面的皮肤。</li>
</ol>9. 血压 Radiotelemeter 的植入
<ol><li>在它的背部重新定位鼠标, 延髓末端指向外科医生。根据需要调整麻醉 nosecone。在这一点上管理第二剂量的甘罗溴铵 (见 7.1.3)。</li>
	<li>用手术刀 (#11) 在颈部区域的皮肤上进行中线切口, 从刚下的动物的下颚开始, 伸展到胸腔上方。将腺组织分开, 以暴露基础颈部肌肉。暴露左颈总动脉并与周围组织分离。 注意: 小心不要损害迷走神经, 因为这会导致手术后死亡率的增加。<ol>
<li>通过三件6-0 丝缝合材料在动脉下面。将一条缝合线尽可能 rostrally, 并将其系在咬合的容器上。在船的长度中间放置第二条缝合线, 并松散地系在一起。将最后缝合的位置尽可能 caudally, 并松散地打结。</li>
		<li>收回延髓-大多数缝合和安全的 nosecone 与一小块脐胶带。收回尾部-大多数缝合与微蚊钳, 以限制血液流动的血管。</li>
		<li>在容器壁上做一个小切口, 用细弹簧剪刀尽可能 rostrally。将小鼠血压 radiotelemeter 导管放入血管内, 并推进骶管缝合。<ol>
<li>配合中间缝合, 暂时稳定导管, 松开骶管并推进导管10毫米. 在导管周围栓缝合以确保到位。</li>
		</ol>
</li>
		<li>将遥测身体沿右侧侧的皮下口袋隧道。</li>
	</ol>
</li>
</ol>10. 颈静脉导管的植入和 Exteriorization
<ol><li>使用小棉尖的喷头暴露右颈静脉。在船上通过两块6-0 丝缝合材料。<ol>
<li>将一条缝合线尽量 rostrally, 并系上咬合。将第二缝合线尽可能 caudally, 并轻轻缩回以阻止血管内的血液流动。</li>
		<li>使用精细的弹簧剪刀, 使一个小切口在血管壁尽可能接近延髓缝合尽可能。Catheterize 静脉与热拉伸油管 (外径1.02 毫米, 伸展到 OD 0.64 毫米), 这是预先填充无菌生理盐水。 注: 确保导管尖端与手术刀切割, 以产生一个圆形的斜面, 以防止血管穿孔。确定导管 (死空间) 中的流体体积以供参考 (参见下面的步骤 14.4-14.6)。<ol>
<li>将导管向前推进8毫米到静脉内。通过将丝缝线绑在血管和导管上, 以及应用一小滴凝胶配方氰胶来保证导管的安全。</li>
		</ol>
</li>
	</ol>
</li>
	<li>将鼠标放在左侧。使用13G 不锈钢针在颈背 midscapular 区内插入静脉导管。</li>
	<li>在他的背部重新定位鼠标。用不连续缝线闭合颈部切口。</li>
	<li>把动物放在俯卧的位置。将一个小的皮下按钮插入静脉导管。用缝线固定在皮肤下的按钮。将相应的不锈钢弹簧螺纹在静脉导管上, 并将其固定在皮肤按钮上以保护导管。</li>
</ol>11. 保护形象化电极引线
<ol><li>安全的聚乙烯基座保护电极导致底层肌肉与组织胶粘剂。在法兰上缝合上覆的皮肤以进一步支撑。</li>
</ol>12. 手术后恢复
<ol><li>将抗生素软膏应用于所有切口。</li>
	<li>管理止痛药。在恢复期内, 如果动物表现出疼痛或窘迫的迹象, 则应根据需要管理额外剂量的止痛药。</li>
	<li>把鼠标放在一个新陈代谢的笼子里, 里面衬着木屑床上用品和纸巾来恢复。持续监测动物, 并不要让它无人看管, 直到它恢复知觉, 并可以维持胸骨卧床。在这一点上介绍环境浓缩和食物和水 (ad 随意)。</li>
	<li>线圈电极引出笼外直到实验的时间。</li>
	<li>把笼子放在温暖的热垫上, 在第一个24小时内恢复。将不锈钢弹簧和静脉导管连接到一个旋转/输液系统, 用于在恢复期内连续注入生理盐水 (0.5 毫升/小时)。</li>
	<li>由于形象化导管和电极引线的性质, 确保动物遗骸单独存放在专用笼中。</li>
</ol>13. 记录血压和北美放射学会的实验装置
<ol><li>用一个简单的法拉第笼装备一个不锈钢顶部防振台。 注意: 这个法拉第笼可以建造一个木框架和铝网网。电接地表/法拉第笼, 以消除任何电气噪音。</li>
	<li>在法拉第笼内放置一个血压 radiotelemetry 接收机。</li>
	<li>将 radiotelemetry 接收器连接到相关的压力输出适配器。将此适配器连接到数据采集系统, 以便在线记录血压。</li>
	<li>焊锡两个女性引脚连接器, 是免费的电极男性引脚连接器 (黄铜镀金) 到两端的配对, 屏蔽聚氯乙烯绝缘电缆。将这一对电缆的两端焊在香蕉插头上。将香蕉插头连接到前置放大 headstage (10X 放大)。</li>
	<li>将此前置放大器连接到差分放大器。调整设置放大到神经信号 x10, 000。按如下方式调整过滤器设置: 低剪切, 100Hz;高切, 1000 赫兹。</li>
	<li>在手术后48至72小时内, 将含有鼠标的主笼放置在法拉第笼内的 radiotelemetry 接收机上。打开 radiotelemetry 探头记录血压信号。 注: 在手术前1周的时间内, 将顺应放置在安装的笼子里是最佳的。</li>
	<li>解开电极引线并将双极电极的针脚连接器插入上述 (13.4) 所述的相应的女性 pin 连接器, 开始记录北美放射学会。</li>
	<li>使用计算机在线显示并同时记录血压信号, 同时注入生理盐水或兴趣解决方案。以每秒2500个样本的最低速率记录数据。</li>
</ol>14. 样品试验协议和北美放射学会信号的验证
<ol><li>确保老鼠在自己的笼子里很舒服, 自由自在地享受食物和水。遵循机构动物护理指南, 以验证正常的外观和行为。</li>
	<li>把老鼠放在温度和湿度控制的房间里, 北美放射学会记录会在那里进行。确保静脉输液继续如上文所述。</li>
	<li>一旦动物位于上面描述的记录设置中, 在记录一个小时的基线血压和北美放射学会数据之前, 至少允许30分钟的稳定。确保动物在录音过程中静静地休息, 因为自然运动与交感神经音调的增加有关。注意当动物在录音时直接在数字跟踪上移动, 这样在分析过程中就会被忽略。</li>
	<li>先缓慢注射硝普钠丸 (2.5 µg/克的体重在25µL 盐水中) 进入输液线, 测试反射反应。用50µL 生理盐水慢慢冲洗线。确保导管的死腔被清除。记录血压和北美放射学会2至5分钟。</li>
	<li>慢慢注射一丸的肾上腺素 (20 µg/克的体重在25µL 生理盐水)。冲洗50µL 生理盐水。确保导管的死腔被清除。记录血压和北美放射学会10至15分钟。</li>
	<li>通过缓慢注射节阻滞剂丸, 六甲铵 (50 µg/g 体重25µL 生理盐水) 进入输液线, 验证神经信号的节后性质。50µL 盐水冲洗。确保导管的死腔被清除。继续录制几分钟。</li>
	<li>使用六甲铵管理后保留的剩余活动作为对北美放射学会分析中使用的背景噪声的估计 (如下所述)。</li>
	<li>弄死小鼠过量的异氟醚 (逐步增加0.5 至 5%), 并继续记录北美放射学会进一步30分钟。注意: 剩余信号也可用作北美放射学会分析的背景噪声估计。</li>
</ol>15. 数据分析
<ol><li>使用数据采集软件分析原始血压和北美放射学会痕迹。<ol>
<li>使用该软件对原始北美放射学会跟踪进行数字集成和全波校正。为积分设置选择 "绝对积分";应用0.1 秒6的时间常数衰减。</li>
		<li>分析实验协议各部分的集成北美放射学会信号 (显示在µV·s 单元中)。当动物发生移动时, 不要理会录音片段。分别对实验的基线和实验部分进行至少3项测量。</li>
		<li>对硝普钠或肾上腺素的最小和最大血压水平进行北美放射学会分析, 分别评估反射敏感性。</li>
		<li>平均为实验性协议的每一部分所做的单个测量结果产生一个值。</li>
		<li>通过计算北美放射学会从基线 (在100%7 中指定) 的百分比变化来量化北美放射学会响应。根据需要完成统计分析。 注: 在本例中, 通过学生的t测试完成了北美放射学会对硝普钠和肾上腺素的反应的统计分析;P值 < 0.05 接受了重要性。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

在这里, 我们概述, 证明和验证了一种新的方法, 目标评估的北美放射学会在有意识的小鼠, 自由移动和休息舒适, 在他们的家庭笼子。术后植入动脉压 radiotelemeter, 留置静脉输液导管和定制设计的双极北美放射学会电极, 小鼠从手术中恢复, 并没有被不受干扰48至72小时。老鼠仍然舒适地定居在他们的笼子里, 在任何时候 (包括试验期), 不受限制地获得食物, 水和环境丰富。所有随后的实验操作由调查?...

随着无数基因工程模型的发展, 对在生物医学研究的几个领域使用小鼠的兴趣继续扩大。在大多数情况下, 技术进步与小鼠在生理学中的使用量的增加保持同步, 现在有一个令人印象深刻的选择, 专门用于测量小鼠重要生理参数的微型设备。尽管在有意识的大鼠中直接测量自主神经音的遥测装置已经使用了十年, 但目前尚无用于评估有意识小鼠神经活动的小型化装置。研究者通常通过评估自主神经系统对间接方法的贡献 (如血浆或尿儿茶酚胺、药理自主阻断、血液形态谱分析) 来规避这一局限性。压力/心率)1。
虽然这些方法提供了有价值的信息, 但结果却是整体自主语气的全球图景, 而不是揭示孤立的神经群对正在调查的现象的离散贡献。另外, 在麻醉小鼠中, 直接记录来自特定神经的活动, 这引起了许多关注。在手术后几个小时内, 在麻醉鼠的生理范围内维持稳定的血压是极其困难的。事实上, 在这些类型的实验中, 血压往往未报告或呈现在极低的水平 (即 60-80 mmHg vs > 100mmHg 在有意识的鼠标)2。在麻醉鼠标制剂中显示的心血管系统的脆弱性往往排除了对自主神经活动的有意义的评估, 因为血压和交感神经张力之间的相互依赖关系3, 4。
为了解决这一局限性, 开发了一种新的方法, 直接记录的肾脏交感神经活动 (北美放射学会) 在有意识的, 无拘无束的小鼠, 不受干扰, 在他们的家庭笼子内。本文详细介绍了该技术成功实施的外科和实验方法。该制剂使研究者可以同时记录动脉压力通过 radiotelemetry 除了北美放射学会, 增加的能力, 静脉注射注入的药物的利益, 而不干扰鼠标。
手术后二十四小时, 小鼠表现正常, 不表现出疼痛或窘迫的迹象。实验记录可以在手术后开始48到72小时, 而老鼠在其家庭笼子里舒适地休息, 不受限制地获得食物、水和环境浓缩。明确的北美放射学会痕迹和这种神经种群对动物正常运动的特征反应 (如进食和梳理) 除了对全身血压的药理调节外, 还表现出来。节封锁进一步验证了北美放射学会信号的质量和特异性。这篇手稿包含了对该技术5最初发布的描述的视听补充。

<table><tbody><tr><td>Teflon-coated stainless steel multiple stranded wire</td><td>A-M Systems</td><td>793200</td><td>0.001in diameter bare; 0.0055in diameter coated</td></tr><tr><td>#11 Scalpel Blade</td><td>Fisher Scientific</td><td>ALMM9011</td><td></td></tr><tr><td>Soldering Iron and solder</td><td></td><td></td><td>Any make or model suitable</td></tr><tr><td>Male miniature pin connectors</td><td>A-M Systems</td><td>520200</td><td>Brass with gold plating</td></tr><tr><td>Female miniature pin connectors</td><td>A-M Systems</td><td>520100</td><td>Brass with gold plating</td></tr><tr><td>Heat Shrink tubing</td><td>Radio Shack</td><td>Model #: 278-1610 |&amp;nbsp;Catalog #: 2781610</td><td>1.6 mm diameter</td></tr><tr><td>Polyethylene 90 (PE90) tubing</td><td>VWR</td><td>CA-63018-703</td><td>0.86mm inner diameter; 1.27mm outer diameter</td></tr><tr><td>Dissecting microscope</td><td>Leica Microsystems</td><td>Leica M80</td><td>Any make or model also suitable</td></tr><tr><td>Polyethylene 10 (PE10) tubing</td><td>Braintree Scientific</td><td>PE10&amp;nbsp;50 FT</td><td>0.28mm inner diameter; 0.61mm outer diameter</td></tr><tr><td>Super Glue Liquid</td><td>Loctite</td><td>n/a</td><td>Liquid Formula; any brand suitable</td></tr><tr><td>Super Glue Gel</td><td>Loctite</td><td>n/a</td><td>Gel Formula; any brand suitable</td></tr><tr><td>Polyethylene tubing</td><td>Scientific Commodities</td><td>BB31695-PE/13</td><td>For pedestal 2.7mm inner diameter; 4.0mm outer diameter</td></tr><tr><td>Hospital Sterilization Services &amp;amp; Ozone Sterilization packets</td><td></td><td></td><td>Contact local hospital sterilization services</td></tr><tr><td>Isoflurane anesthesia</td><td>Abbott</td><td>05260-05</td><td></td></tr><tr><td>Deltaphase isothermal heat pads &amp;amp; surgical table</td><td>Braintree Scientific</td><td>39OP</td><td>Keep heat pads warm in a 37&amp;deg;C water bath; Corresponding surgical table essential</td></tr><tr><td>Glycopyrrolate</td><td>Amdipharm Mercury Company Limited</td><td>n/a</td><td></td></tr><tr><td>Isoflurane vaporizer system &amp;amp; flow gauge</td><td>Braintree Scientific</td><td>VP I</td><td>Include medical grade oxygen supply</td></tr><tr><td>Tissue scissors</td><td>Fine Science Tools</td><td>14173-12</td><td></td></tr><tr><td>Fine spring scissors</td><td>Fine Science Tools</td><td>15006-09</td><td></td></tr><tr><td>Small cotton-tipped applicators</td><td>Fisher Scientific</td><td>23400100</td><td></td></tr><tr><td>Fine Straight Forceps</td><td>Fine Science Tools</td><td>11254-20</td><td>#5, FST by Dumont Biologie Tip</td></tr><tr><td>Angled Forceps</td><td>Fine Science Tools</td><td>11251-35</td><td>#5/45 FST by Dumont</td></tr><tr><td>Small Absorbent Spears</td><td>Fine Science Tools</td><td>18105-03</td><td></td></tr><tr><td>Parafilm</td><td>Sigma Aldrich</td><td>BR701605&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>Kwik-Sil 2 component Silicone Polymer</td><td>World Precision Instruments (WPI)</td><td>KWIK-SIL</td><td>Purchase extra specialized tips from WPI</td></tr><tr><td>5-0 Polysorb Suture</td><td>Tyco Healthcare</td><td>n/a</td><td></td></tr><tr><td>6-0 Silk Suture</td><td>Braintree Scientific</td><td>SUT-S&amp;nbsp;104</td><td>Deknatel brand, spool</td></tr><tr><td>Radiotelemetry Probe</td><td>Data Sciences International (DSI)</td><td>TA11-PAC10</td><td></td></tr><tr><td>Radiotelemetry Receiver</td><td>Data Sciences International (DSI)</td><td>PhysioTel RPC-1</td><td></td></tr><tr><td>Ambient Pressure Reference</td><td>Data Sciences International (DSI)</td><td>Apr-01</td><td></td></tr><tr><td>Pressure Output Adapter</td><td>Data Sciences International (DSI)</td><td>R11CPA</td><td></td></tr><tr><td>Rena Pulse Tubing</td><td>Braintree Scientific</td><td>RPT-040</td><td></td></tr><tr><td>Infusion Swivel</td><td>Instech Solomon</td><td>375/D/22</td><td></td></tr><tr><td>Swivel Support Arm &amp;amp; Mount</td><td>Instech Solomon</td><td>SMCLA</td><td></td></tr><tr><td>Polysulfone button&amp;nbsp;</td><td>Instech Solomon</td><td>LW62S/6</td><td></td></tr><tr><td>Stainless steel spring</td><td>Instech Solomon</td><td>PS62</td><td></td></tr><tr><td>Vetbond surgical adhesive</td><td>3M</td><td>n/a</td><td></td></tr><tr><td>Triple Antibiotic Ointment</td><td>Fougera</td><td>n/a</td><td></td></tr><tr><td>PowerLab 8 Channel Data Acquisition System &amp;amp; Software</td><td>ADInstruments</td><td>PowerLab 8/35</td><td></td></tr><tr><td>PVC Insulated Cable</td><td>Belden</td><td>PVC Audio Connection Cable 32 AWG</td><td></td></tr><tr><td>Preamplification Headstage</td><td>Dagan Corporation</td><td>Model 4002</td><td></td></tr><tr><td>Differential Amplifier</td><td>Dagan Corporation</td><td>EX4-400</td><td></td></tr><tr><td>Sodium Nitroprusside</td><td>Sigma Aldrich</td><td>71778-25G</td><td></td></tr><tr><td>Phenylephrine</td><td>Sigma Aldrich</td><td>P6126-5G</td><td></td></tr><tr><td>Sterile Physiological Saline 0.9% NaCl</td><td>Beckton Dickinson</td><td></td><td>Contact local hospital supplier</td></tr><tr><td>hexamethonium</td><td>Sigma Aldrich</td><td>H0879-5G</td><td></td></tr><tr><td>Stainless Steel top anti vibration table</td><td>n/a</td><td>n/a</td><td>Custom designed in-house; Solid steel plate on a benchtop is also suitable</td></tr><tr><td>Faraday cage</td><td>n/a</td><td>n/a</td><td>Custom designed and constructed in-house</td></tr><tr><td>Small animal hair trimmer</td><td>n/a</td><td>n/a</td><td>Drugstore, men&#039;s beard trimmer suitable</td></tr><tr><td>Dipilatory Cream</td><td>n/a</td><td>n/a</td><td>Veet brand, sensitive skin formula</td></tr><tr><td>10% Povidone Iodine</td><td>Purdue Products</td><td>Betadiene</td><td></td></tr><tr><td>70% Ethanol</td><td>n/a</td><td>n/a</td><td></td></tr><tr><td>Steel microretractors</td><td>n/a</td><td>n/a</td><td>Made in-house. Bend a steel paper clip &amp;amp; loop 4-0 silk to form a retractor</td></tr><tr><td>Hemostats</td><td>Fine Science Tools</td><td>13011-12</td><td></td></tr><tr><td>Heat Gun</td><td>Fisher Scientific</td><td>09-201-27</td><td></td></tr></tbody></table>

novel approach for simultaneous recording of renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious, unrestrained mice.

肾脏交感神经对生理和病理生理学都有显著的促进作用。评价肾交感神经活动 (北美放射学会) 对慢性肾脏疾病、高血压、心力衰竭、糖尿病和肥胖等许多研究领域都有很大的兴趣。因此, 明确评估交感神经系统的作用是正确解释实验结果和了解疾病过程的必要条件。传统上, 北美放射学会被麻醉的啮齿类动物所测量, 包括老鼠。然而, 小鼠在麻醉和手术中通常表现出很低的系统性血压和血流动力学不稳定。鉴于交感神经张力与心血管状态的密切关系, 北美放射学会的意义解释被这种非生理状态所迷惑。为了解决这种传统方法的局限性, 我们开发了一种新的方法来测量有意识的、自由移动的小鼠北美放射学会。对小鼠进行长期的 telemeters 检测, 以持续监测血压以及颈静脉输液导管和定制设计的双极性电极直接记录北美放射学会。在48-72 小时的恢复期后, 存活率为 100%, 所有小鼠正常表现。在这个时间点, 北美放射学会成功地记录在80% 只老鼠身上, 在70% 和50% 的小鼠中, 有可行的信号可以获得4和5天的手术后。所有小鼠 (116±2 mmHg; n=10) 均记录生理血压。记录北美放射学会增加与饮食和梳理, 在文献中建立。此外, 北美放射学会的节阻断和调节血压与药理剂。本文介绍了一种有效的、可管理的北美放射学会在有意识、自由移动的小鼠中清晰记录的方法。

经描述的协议, 生存率为 100%-所有的小鼠在这项研究中幸存下来, 并恢复良好的手术过程后。在24小时的手术准备, 所有的老鼠表现正常, 展示典型的饮食, 梳理和探索行为。在这个时候, 没有动物显示出任何疼痛或痛苦的迹象。术后48小时, 在12只老鼠中, 有一个可核实和清晰的北美放射学会信号记录在10。这个信号在这些老鼠被维护了72小时后手术, 然而一个真正的北美放射学...

Watch this Scientific Journal Video about Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice. at

Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

麻醉小鼠表现为非生理系统血压, 由于血压和自主神经系统之间的密切关系, 导致对自主语气有意义的评估。为此, 本文概述了在有意识小鼠中, 用静脉输液同时记录肾交感神经活动和血压的新方法。

清醒、无拘无束的小鼠静脉输液同时记录肾交感神经活动和血压的新方法。

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity ...

novel-approach-for-simultaneous-recording-renal-sympathetic-nerve

Research

JoVE Journal

Medicine

10.1K 次观看.  University of Alberta. 麻醉小鼠表现为非生理系统血压, 由于血压和自主神经系统之间的密切关系, 导致对自主语气有意义的评估。为此, 本文概述了在有意识小鼠中, 用静脉输液同时记录肾交感神经活动和血压的新方法。

视频: Novel Approach for Simultaneous Recording of Renal Sympathetic Nerve Activity and Blood Pressure with Intravenous Infusion in Conscious, Unrestrained Mice.

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

在自觉的，奔放的小鼠正常血糖高胰岛素 - 夹钳

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

科研

医学

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

股动脉和静脉导管采血，药品监督管理局和意识的血压和心率测量

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

Transcutaneous Assessment of Renal Function in Conscious Rodents

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and time-consuming. In addition, serial blood or urine samples are required, with the associated stress for the experimental animals. A recent technique significantly reduces the investment in time and resources, minimizing the invasiveness and the animal stress, but being equally valid as the traditional approaches. The method measures transcutaneously renal function. Using an optical device and the exogenous renal marker fluorescein isothiocyanate (FITC)-sinistrin, this technique is capable of measuring the elimination kinetics of the marker through the skin. With neither blood nor urine samples nor the associated laboratory assays needed, the results of the transcutaneous measurement are almost instantaneously available. The method has been already validated in different species and successfully applied in several models of renal pathology. Moreover, due to its minimally invasive characteristics, it is suitable for sequential measurements within the same animal. Here is provided a detailed protocol to carry out the transcutaneous assessment of renal function in rodents.

Determination of glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional procedures to measure this parameter are cumbersome and require a large investment of time. Here we describe a faster and minimally invasive method to determinate GFR transcutaneously.

在鼠害自觉肾功能的评估皮

Glomerular filtration rate (GFR) is the gold standard to assess overall kidney function. However, traditional methods to evaluate GFR are cumbersome and ...

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are limited resources directing scientists in the techniques for measuring and analyzing these variables. This protocol describes the methods for measuring RSNA and mean arterial pressure in anesthetized rats. The protocol also includes the approaches for accessing the brain during RSNA recordings for central nervous system (CNS) manipulations. The RSNA recording technique is compatible with pharmacologic, optogenetic, or electrical stimulation of the CNS. The approach is useful when an investigator will measure short-term (min to h) autonomic responses in non-survival experiments to correlate anatomically with CNS nuclei. The approach is not intended to be used to obtain chronic (survival) recordings of RSNA in rats. Discharges in RSNA, averaged rectified RSNA, and mean arterial pressure can be quantified and analyzed further using parametric statistical tests. Methods for obtaining venous access, recording mean arterial pressure telemetrically, and brain fixation for future histological analysis are also described in the article.

Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

麻醉大鼠中枢神经系统操作对肾交感神经活动的急性变化量化

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...

神经科学

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

大鼠同时心电图记录及侵入性血压测量

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

Implantation of Combined Telemetric ECG and Blood Pressure Transmitters to Determine Spontaneous Baroreflex Sensitivity in Conscious Mice

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. The baroreflex is a key homeostatic mechanism to counteract acute, short-term changes in arterial BP and to maintain BP in a relatively narrow physiological range. BP is sensed by baroreceptors located in the aortic arch and carotid sinus. When BP changes, signals are transmitted to the central nervous system and are then communicated to the parasympathetic and sympathetic branches of the autonomic nervous system to adjust HR. A rise in BP causes a reflex decrease in HR, a drop in BP causes a reflex increase in HR.
Baroreflex sensitivity (BRS) is the quantitative relationship between changes in arterial BP and corresponding changes in HR. Cardiovascular diseases are often associated with impaired baroreflex function. In various studies reduced BRS has been reported in e.g., heart failure, myocardial infarction, or coronary artery disease. 
Determination of BRS requires information from both BP and HR, which can be recorded simultaneously using telemetric devices. The surgical procedure is described beginning with the insertion of the pressure sensor into the left carotid artery and positioning of its tip in the aortic arch to monitor arterial pressure followed by the subcutaneous placement of the transmitter and ECG electrodes. We also describe postoperative intensive care and analgesic management. After a two-week period of post-surgery recovery long-term ECG and BP recordings are performed in conscious and unrestrained mice. Finally, we include examples of high-quality recordings and the analysis of spontaneous baroreceptor sensitivity using the sequence method.

The baroreflex is a heart-rate regulation mechanism by the autonomic nervous system in response to blood-pressure changes. We describe a surgical technique to implant telemetry transmitters for continuous and simultaneous measurement of electrocardiogram and blood pressure in mice. This can determine spontaneous baroreflex sensitivity, an important prognostic marker for cardiovascular disease.

植入遥测心电图和血压递质组合以确定意识小鼠的自发压力反射敏感性

Blood pressure (BP) and heart rate (HR) are both controlled by the autonomic nervous system (ANS) and are closely intertwined due to reflex mechanisms. ...

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital functions of filtration and metabolism in kidney function. Many acute studies have been carried out in anesthetized animals to measure RBF under various conditions to determine mechanisms responsible for the regulation of kidney perfusion. However, for technical reasons, it has not been possible to measure RBF continuously (24 h/day) in unrestrained unanesthetized rats over prolonged periods. These methods allow the continuous determination of RBF over many weeks while also simultaneously recording blood pressure (BP) with implanted catheters (fluid-filled or by telemetry). RBF monitoring is carried out with rats placed in a circular servo-controlled rat cage that enables the unrestrained movement of the rat throughout the study. At the same time, the tangling of cables from the flow probe and arterial catheters is prevented. Rats are first instrumented with an ultrasonic flow probe placement on the left renal artery and an arterial catheter implanted in the right femoral artery. These are routed subcutaneously to the nape of the neck, and connected to the flowmeter and pressure transducer, respectively, to measure RBF and BP. Following surgical implantation, rats are immediately placed in the cage to recover for at least one week and stabilize the ultrasonic probe recordings. Urine collection is also feasible in this system. The surgical and post-surgical procedures for continuous monitoring are demonstrated in this protocol.

The present protocol describes a long-term continuous measurement of renal blood flow in conscious rats and simultaneously recording blood pressure with implanted catheters (fluid-filled or by telemetry).

意识大鼠肾血流量的长期连续测量

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital ...

<meta charset="utf8"></head><body>小鼠深部脑干结构的倾斜硅多电极记录

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many brain regions. However, recording neuronal activity from deep caudal structures like the brainstem using multi-channel probes remains challenging. A significant concern is finding a trajectory for probe insertion that avoids large blood vessels, such as the superior sagittal venous sinus and the transverse venous sinus. Injuring these large veins can cause extensive bleeding, damage to the underlying brain tissue, and potentially death. This approach describes targeting brainstem structures by coupling anterior coordinates with an angled approach, allowing the recording probe to penetrate the brain below high-risk vascular structures. Compared to a strictly vertical approach, the angled approach maximizes the number of brain regions that can be targeted. Using this strategy, the ventrolateral periaqueductal gray (vlPAG), a brainstem region associated with REM sleep, can be reproducibly and reliably accessed to obtain single-unit, multi-electrode recordings in head-fixed mice before and during sevoflurane anesthesia. The ability to record neuronal activity in the vlPAG and surrounding nuclei with high temporal resolution is a step forward in advancing the understanding of the relationship between REM sleep and anesthesia.

<meta charset="utf8"></head><body>作者聚焦：研究小鼠麻醉诱导的睡眠通路和神经元兴奋性

This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

来自头部固定小鼠脑干的急性单单位多电极记录

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many ...

清醒、无拘无束的小鼠静脉输液同时记录肾交感神经活动和血压的新方法。

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在自觉的，奔放的小鼠正常血糖高胰岛素 - 夹钳

股动脉和静脉导管采血，药品监督管理局和意识的血压和心率测量

在鼠害自觉肾功能的评估皮

麻醉大鼠中枢神经系统操作对肾交感神经活动的急性变化量化

大鼠同时心电图记录及侵入性血压测量

植入遥测心电图和血压递质组合以确定意识小鼠的自发压力反射敏感性

意识大鼠肾血流量的长期连续测量

来自头部固定小鼠脑干的急性单单位多电极记录

来自头部固定小鼠脑干的急性单单位多电极记录

来自头部固定小鼠脑干的急性单单位多电极记录