这项研究得到了国立护理研究所 (K99/R00NR014369) 的支持。

所描述的所有方法都是由芝加哥伊利诺伊大学的机构动物护理委员会批准的。
1. 创建双极北美放射学会电极
<ol><li>要制造电极, 切两件不锈钢丝每约18毫米长。切割一条聚乙烯 (PE-50) 油管约15毫米长。将两条导线进油管, 使导线两端突出。</li>
	<li>从导线的两端除去绝缘物;修剪电线留下2-3 毫米裸露的电线。一端, 在暴露的导线上卷曲男性针脚。将针脚安全地焊接到导线上, 确保连接器条内的针脚牢固, 并覆盖与环氧树脂的连接。 注意: 避免焊接的另一种方法是使用快速连接/释放鳄鱼夹子。</li>
	<li>在电极的另一端, 从导线的两端除去绝缘, 留下2-3 毫米暴露的导线。弯曲这部分电线, 以创建小 "V" 形钩在绝缘线。 注: 这是电极的一部分, 将与肾交感神经接触。密封这一端以防止流体进入油管是很重要的。有机硅或环氧树脂有效地可以使用。</li>
</ol>2. 管理麻醉和准备手术场所
<ol><li>对雄性大鼠进行麻醉 (年龄9-11 周, 重150-400 克)。通过腹腔 (IP) 注射, 管理戊巴比妥钠50毫克/千克。为了评估手术中的麻醉稳定面, 每15分钟检查一次脚趾捏反射, 并根据需要重新进行麻醉。 注: 戊巴比妥钠 (戊巴比妥) 用于以前的研究, 以达到持续的麻醉平面, 不干扰北美放射学会4,5,6的调制。该协议是为非生存手术, 因此没有恢复/术后监测期。</li>
	<li>根据机构动物护理指导方针 (i.) 准备手术地点; 剃掉老鼠的腹部、背部和头部; 用10% 聚维酮碘溶液清洗皮肤; 涂抹眼部润滑剂; 把老鼠放在暖气垫上)。在实验期间保持体温在37摄氏度。</li>
</ol>3. Cannulate 股静脉 (供静脉进入)
<ol><li>添加肝素到0.9% 无菌生理盐水 (达到20单位/毫升)。用22G 针将血气盐水填充1毫升注射器。将15厘米的 PE-50 油管连接到针上, 并用溶液填充油管。</li>
	<li>与鼠仰卧卧, 创建一个2厘米水平切口通过腹股沟区。采用棉尖涂抹器, 解剖结缔组织, 露出股静脉和动脉。为保持切口开放, 无论是应用单钩弹性手术留在外科领域与丝绸胶带或使用小止血剂。</li>
	<li>使用止血剂在90°角度弯曲22G 针的尖端, 作为导管介绍人10。</li>
	<li>显微镜下的血管可视化。用弯钳轻轻地将静脉和动脉分开。在静脉下放置两个12厘米长的5-0 丝缝合线 (一个远端和一个近端);用同样的方式把缝线放在动脉下面。<ol>
<li>将远端 (底) 缝线系在静脉咬合;使用丝带或小止血剂将此缝合线的边缘固定在手术领域。轻轻拉紧静脉, 但不要用这么多的力量, 船只会撕裂。将静脉垂直于外科医生的显性手。</li>
	</ol>
</li>
	<li>在近端缝合中使用松动的半结, 简单咬合静脉。用细腻的止血钳, 轻轻夹住这缝合咬合血流。将22G 针与弯曲尖端握在非显性手上;使用主手将油管与镊子扣紧。</li>
	<li>用导管介绍人 (步骤 3.3) 在静脉穿刺一个小孔, 并将 PE-50 导管 (预填充血气生理盐水) 插入容器内;使用弯曲的针保持打开的容器开放, 并协助将导管的尖端定位到容器10。<ol>
<li>释放近端缝合, 轻轻冲洗0.2 毫升的血气生理盐水进入静脉;提前导管。检查血液从静脉回流, 以确保适当的放置。完成近端结, 并与远端缝合领带, 确保静脉内的导管。</li>
	</ol>
</li>
	<li>在实验中使用静脉通路来管理补充麻醉或药物和收集血液。如果需要定期进行静脉输液和血液取样, 请合并3路连接器。每15分钟检查一次有害的脚趾捏反射, 滴定麻醉。</li>
</ol>4. Cannulate 股动脉用于平均动脉压力监测
<ol><li>显微镜下的动脉可视化。类似于静脉插管的方法, 系远端缝合 (步骤 3.4) 咬合动脉;将此缝合线的边缘固定在带丝胶带的外科手术场上, 并将动脉垂直于外科医生的主导手。</li>
	<li>使用压力传感器/输液系统时的动脉通路<ol>
<li>将压力传感器/油管连接至500毫升0.9% 盐水袋。用盐水冲洗油管, 除去所有气泡, 将袋子放在压力诱导袋内加压系统。</li>
		<li>如步骤3.1 所述, 通过22G 针填充1毫升注射器和血气盐水, 并将15厘米的 PE-50 连接到针 (冲洗油管与血气生理盐水)。</li>
		<li>在近端缝合中使用松散的半结来短暂咬合动脉。将导管介绍人 (步骤 3.3) 放在非显性手上;用血管插管钳将 PE-50 的远端放在占主导地位的手上。用弯曲的22G 针在动脉内穿刺一个孔, 并将套管插入血管内。</li>
		<li>释放近端缝合, 轻轻冲洗0.2 毫升的血气生理盐水进入动脉, 尽量提前导管。检查动脉血返回以确保正确放置。完成近端结, 并与远端缝合领带, 确保导管内动脉。将动脉线连接到压力传感器/油管。 注: 导管远端部分可贴在大鼠后肢上, 以保证动脉线的安全。另一种方法的容器插管是描述的 Jespersen et al。11他们的协议不同于使用拉钩传播切口, 胶-而不是缝合-安全油管, 并且方法不包括弯曲的针介绍人。</li>
	</ol>
</li>
	<li>使用遥测的动脉通路<ol>
<li>在动脉插管前, 在高放大倍数下检查压力传感导管。确保导管没有气泡/碎片, 并有一个完整的半月板之间的液体填充 (近端) 和凝胶填充 (远端) 成分。在每次植入之前, 将凝胶在导管的远端端重新填充。用磁铁打开发射机;在手术中监测 BP, 以承受完美的安置。</li>
		<li>在近端缝合中使用松动的半结, 短暂咬合股动脉。将导管介绍人 (步骤 3.3) 放在非显性手上。用导管插管钳握住遥测装置套管的尖端, 避免从尖端中置换凝胶。</li>
		<li>用弯曲的22G 针在动脉内穿刺一个孔, 用导管插管钳将套管插入动脉, 以避免从尖端中置换凝胶。尽量提前插管。使用近端和远端缝合连接, 确保压力导管的安全。</li>
		<li>将遥测植入物的身体塞在切口附近的侧面, 并用4-0 尼龙缝线缝合切割针。在记录期结束时, 通过磁铁将遥测装置关闭, 以节省电池寿命。</li>
	</ol>
</li>
</ol>5. 将大鼠定位在立体定向手术框架中以进入大脑
<ol><li>将大鼠轻轻地移动到立体定向手术框架中的俯卧位。</li>
	<li>将大鼠放置在耳棒之间, 调整切牙条以平衡 lambda 和 bregma 的高度。定位可能取决于大鼠的应变、体重和中枢神经系统目标的位置。</li>
	<li>通过头皮中线做一个2厘米 rostrocaudal 手术刀切口。使用棉尖的喷头, 牢固地去除头骨表面的结缔组织。将过氧化氢应用于颅骨, 以帮助可视化 bregma、lambda 和中线缝合。</li>
	<li>使用大鼠大脑图谱来指导目标12, 钻一个毛刺孔截骨, 大小为电极进入, 通过头骨。</li>
</ol>6. 隔离肾交感神经
<ol><li>将导线北美放射学会电极 (步骤 1.1-1.3) 连接到10X 预放大器和微电极放大器。</li>
	<li>将肾神经通过腹膜后切口隔离, 或后, 将大鼠固定在立体定向的框架内。一旦大鼠在立体定向的框架内, 定位北美放射学会电极。使手术刀切开从4-5 厘米以下的肋骨在尾部方向, 稍侧向脊柱。钝解剖切口, 可视化椎旁肌肉。</li>
	<li>用剪刀做一个非常肤浅的1-2 厘米 rostrocaudal 切口在那里脂肪满足肌肉。使用棉尖的喷头, 将脂肪从肌肉中传播到想象肾脏。重要的是不要进入腹膜的空间。</li>
	<li>使用拉钩轻轻地将肾脏与椎旁肌肉分开, 以可视化肾动脉和腹主动脉。不要过度伸展血管, 以免损害肾脏神经。使用 2 "x 2" 棉纱布垫浸泡在盐水中, 以保护肾脏免受伤害。</li>
	<li>在高放大倍数下, 识别切口口袋中的肾神经。神经束最容易在由主动脉和肾动脉形成的直角上可见。肾神经密切跟随肾动脉从主动脉到肾脏。</li>
	<li>选择将放置在记录电极上的神经束的一段。用显微解剖镊子轻轻地解剖周围组织/血管的神经纤维。</li>
	<li>将导线北美放射学会电极固定在支架上 (例如, 附着在支撑架上的鳄鱼夹子)。将电极降低到神经段的水平。用神经钩轻轻地将肾神经段抬到电极上, 而不伸展神经。 注: 神经应在绝缘线的两个 "V" 形钩内休息, 并与神经平行。电极线不能接触任何其他组织, 血液, 或淋巴液。</li>
	<li>用矿物油填充切口, 防止暴露的肾交感神经变得干燥。在切口的皮肤上使用一个末端的接地夹, 另一端附着在法拉第笼上。</li>
	<li>使用高和低通滤波 (10 赫兹和3赫) 将信号定向到放大器。调整增益高达 10 k 包括一个音频显示器, 以评估爆破模式的北美放射学会。使用采样率, 范围介于 200万赫兹 4、5、6、7、8之间。假设 CNS 操作被假定为快速/简短的交感神经应答, 请使用增加的采样速率。</li>
</ol>7. 记录数据
<ol><li>通过反射注射1毫升生理盐水或10µg/毫升肾上腺素 (0.1 毫升) 静脉注射, 评估北美放射学会记录的质量。如图 1所示, 输液应增加 BP 并抑制北美放射学会。平均动脉压力增加 60-80 mmHg 是足够的肾 sympathoinhibition4,13,14。</li>
	<li>如果需要, 调整电极位置以改善信号。如果神经不接触电极上的两个钩子或任何组织、血液或淋巴液与导线接触, 则需要重新定位。 注意: 重新定位的需要是基于神经放电的听觉特征。<ol>
<li>如果北美放射学会的爆发不是周期性地与心脏循环发生, 如果有任何干扰的录音, 然后小心地重新定位电极。</li>
		<li>呼吸运动也会影响北美放射学会记录的质量, 通过将电极轻轻地移动到肌肉运动不会在呼吸中扰乱电极的位置来改善信号。</li>
	</ol>
</li>
	<li>一旦获得清晰的信号, 通过提取矿物油和应用硅胶来覆盖切口口袋中的神经/电极连接, 确保北美放射学会电极到位。在凝胶完全设置之前不要移动老鼠。</li>
	<li>在连续记录北美放射学会和平均动脉压力的同时执行 CNS 操作协议。如果 microinjector/脉冲发生器用于脑干操作, 则可以将该装置的逻辑信号引入北美放射学会/BP 记录, 以记录中枢神经系统操作的时间。</li>
	<li>实验完成后, 通过粉碎硅胶与脊柱肌肉之间记录电极的神经, 确定噪声水平。记录至少三十年代的这个 "零" 值为北美放射学会4,5,6。作为量化噪声的另一种方法, 管理一个短作用的节阻滞剂, 如阿托品, 六甲铵, chlorisondamine, 或 pentolinium 酒石酸8,15,16,17。</li>
	<li>小心地取出北美放射学会电极, 从电线电极上取出硅胶的任何痕迹。保存电极以供重新使用。关闭遥测发射机并取出, 注意不要损坏导管的尖端。</li>
</ol>8. 安乐死 (Transcardiac 灌注)
<ol><li>通过注射染料或荧光、产生电解损伤或通过检测 c-fos 表达来识别中枢神经系统操作的位置。</li>
	<li>如果大脑分析需要固定, 准备 transcardiac 灌注大鼠。评估脚趾捏反射, 以确保大鼠仍然深麻醉。必要时提供补充麻醉。在通风罩中进行多聚甲醛固定剂的 transcardiac 灌注。 注意: 狂热的皮肤/眼部刺激性。</li>
	<li>将油管插入灌注泵中, 并以0.9% 生理盐水为质数。</li>
	<li>5-6 厘米侧切口通过皮肤和腹壁立即下肋骨笼和打开胸腔。小心地将肝脏与隔膜分开。用弯曲钝剪刀在隔膜上做一个小切口。将0.1 毫升肝素直接注入左心室。</li>
	<li>通过一个灌注针进入左心室 (一个不锈钢饲针工作很好的这一步), 要么刺穿心脏或切割一个小切口使用锋利的剪刀, 通过饲针通过, 所以尖端是可见的通过主动脉壁 (但不应达到主弓)。使用手术或电子夹子固定针到位。</li>
	<li>使用输液泵, 管理0.9% 正常生理盐水 (室温)。立即创建一个2-3 毫米切口在右心房创建一个出口盐水冲洗。不要切断降主动脉。继续盐水冲洗, 直到肝脏改变颜色从红色或褐色到淡黄色, 大约400毫升的输液2-3 分钟。</li>
	<li>停止泵。将灌流液切换至固定器 (e, 10% 福尔马林或4% 多聚甲醛);注入400毫升超过2-3 分钟. 移除大脑并在固定溶液中保存标本在4摄氏度过夜, 然后将组织转移到30% 蔗糖 (30 毫升磷酸酯缓冲盐水中溶解的毫升蔗糖) 至少100天或直到大脑下沉, 用于抗冻前 cryogenetic 切片18。</li>
</ol>9. 分析数据
<ol><li>全波矫正原北美放射学会以获得绝对值。全波矫正十年代段的原始噪声信号。重要的是排除任何受低信噪比影响的研究。在量化北美放射学会的研究中, 调查人员采用了先验标准, 如要求信噪比超过2:1 至 6:117、19、20。</li>
	<li>计算非重叠段 (µV) 的平均整流北美放射学会, 并减去噪声估计 (µV)。根据实验的目的, 调查人员可以选择时间间隔, 如十年代 (图 1) 或1秒. 通过使用生理学软件中的波形分析选项或将数据导出到电子表格中计算方法来计算平均值选定的时间间隔。</li>
	<li>为了在不同的动物之间正常化, 表达价值作为进一步分析的百分比变化从基线。使用参数统计进行分组比较。</li>
</ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+renal+nerve+activity+responses+to+baroreceptor+denervation+in+hypertensive+rats.">Dynamic analysis of renal nerve activity responses to baroreceptor denervation in hypertensive rats.</a> Hypertension. 37 (4), 1153-1163 (2001).</li><li>Kunitake, T., Kannan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discharge+pattern+of+renal+sympathetic+nerve+activity+in+the+conscious+rat:+spectral+analysis+of+integrated+activity.">Discharge pattern of renal sympathetic nerve activity in the conscious rat: spectral analysis of integrated activity.</a> Journal of Neurophysiology. 84 (6), 2859-2867 (2000).</li><li>Machado, B. H., Bonagamba, L. 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E., McCarthy, J. J., Stocker, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamatergic+receptor+activation+in+the+rostral+ventrolateral+medulla+mediates+the+sympathoexcitatory+response+to+hyperinsulinemia.">Glutamatergic receptor activation in the rostral ventrolateral medulla mediates the sympathoexcitatory response to hyperinsulinemia.</a> Hypertension. 55 (2), 284-290 (2010).</li><li>Brozoski, D. T., Dean, C., Hopp, F. A., Seagard, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+blockade+of+endocannabinoids+in+the+NTS+modulates+baroreflex-evoked+sympathoinhibition.">Uptake blockade of endocannabinoids in the NTS modulates baroreflex-evoked sympathoinhibition.</a> Brain Research. 1059 (2), 197-202 (2005).</li><li>Barman, S. 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安妮. 芬克是国际数据科学客户咨询委员会的成员。

测量北美放射学会的关键步骤包括: (1) 在将肾脏与椎旁肌分离时避免肾动脉和神经的伸展, 以及将神经段放在记录电极上时 (2) 仔细解剖肾神经纤维从周围组织/血管, (3) 确保电极线没有组织, 血液, 或淋巴液, (4) 防止神经干燥, 通过应用矿物油的肾神经和硅胶的神经电极单位。为了进行故障排除, 必须确保记录系统充分接地。为了获得清晰的北美放射学会信号, 在嵌入硅胶之前, 可以在可视化和侦听?...

关于心血管系统自主控制的临床前发现告知管理疾病的策略, 如高血压、心力衰竭和慢性肾脏疾病。交感神经系统的过度活动和减少的迷走神经心脏口气贡献高血压 (BP)1。慢性升高的肾交感神经流出增强儿茶酚胺分泌, 减少肾血流量, 对心血管/肾脏系统2,3有害后果。为了定义导致自主功能障碍的神经生物学途径, 啮齿类动物的研究对于确定中枢神经系统 (CNS) 神经元调节交感神经参数是很重要的。本议定书的目的是提供有关测量肾交感神经活动 ( 北美放射学会 ) 和 BP 的技术信息 , 并概述在麻醉大鼠中枢神经系统操作中对急感神经变化进行量化的方法。
急性 (非生存) 北美放射学会测量 (持久极小到 h) 是有用的, 当科学家将探测中枢神经系统药理, 电, 或 optogenetically 麻醉大鼠, 以确定特定核的功能。利用这些方法, 研究了孤立核、periaqueductal 灰、pedunculopontine tegmentum 和延髓延髓髓质等结构, 确定了调节交感神经参数4的神经生物学通路, 5,6,7。这一方法对于确定在慢性自主功能障碍8、9的慢性病模型中进一步研究中枢神经系统目标是很重要的。为了完成这些实验, 实验室需要一个焊接铁, 外科显微镜, 立体定向框架, 微电极放大器, 和音频显示器。根据实验室中存在的影响电子噪音的因素, 手术/录音区域可能需要法拉第笼/接地带, 以减少北美放射学会记录中的电噪声。如果大脑分析需要组织固定, 需要一个灌注泵和通风罩。数据可以用多种生理学软件/数据采集 (模拟数字转换器) 单元4、5进行数字化和记录, 并采用不同的分析方法和兼容性, 用于合并遥测信号。.

<table><tbody><tr><td>Stainless steel wire</td><td>A-M Systems; Sequim, WA</td><td>791000</td><td>RSNA electrode</td></tr><tr><td>Polyethylene (PE-50) tubing</td><td>VWR; Radnor, PA</td><td>63019-048</td><td>RSNA electrode; vessel cannulation</td></tr><tr><td>Miniature pin connector</td><td>A-M Systems; Sequim, WA</td><td>520200</td><td>RSNA electrode</td></tr><tr><td>Crimping tool</td><td>Daniels Manufacturing Corp.; Orlando, FL</td><td>M22520</td><td>RSNA electrode</td></tr><tr><td>Connector strip</td><td>Amphenol; Clinton Township, MI</td><td>221-2653</td><td>RSNA electrode</td></tr><tr><td>J-B Kwik Epoxy</td><td>J-B Weld, Sulphur Springs, TX</td><td>8270</td><td>RSNA electrode</td></tr><tr><td>Silicone</td><td>Permatex; Hartford, CT</td><td>2222</td><td>RSNA electrode</td></tr><tr><td>Heparin sodium; Injectable (10 mL vial, 1000 U/mL)</td><td>KV Veterinary Supply; David City, NE</td><td>P03466</td><td>Venous line patency</td></tr><tr><td>Phenylephrine HCl; Injectable (1 mL vial; 10 mg/mL)</td><td>ACE Surgical Supply; Brockton, MA</td><td>950-6312</td><td>Testing renal sympathoinhibition</td></tr><tr><td>Single-hook elastic surgical stays</td><td>Harvard Apparatus; Holliston, MA</td><td>72-2595</td><td>Incision</td></tr><tr><td>Silk surgical tape</td><td>3M, Minneapolis, MN</td><td>1538-0</td><td>Secure surgical stays</td></tr><tr><td>Needles, 20 G</td><td>Sigma-Aldrich; St. Louis, MO</td><td>Z192554-100EA</td><td>Vessel cannulation</td></tr><tr><td>Dumont #7 curved forceps</td><td>Fine Science Tools; Foster City, CA</td><td>11274-20</td><td>Vessel cannulation</td></tr><tr><td>5-0 silk suture ties</td><td>Braintree Scientific; Braintree, MA</td><td>SUT-S 106</td><td>Vessel cannulation</td></tr><tr><td>Delicate hemostatic forceps</td><td>Roboz Surgical Instrument Co.; Gaithersburg, MD</td><td>RS-7117</td><td>Vessel cannulation and RSNA surgery</td></tr><tr><td>Crile Hemostatic forceps</td><td>Fine Science Tools; Foster City, CA</td><td>13004-14</td><td>Needle bending</td></tr><tr><td>Telemetry transmitter</td><td>Data Sciences International; Minneapolis, MN</td><td>PA-10</td><td>Mean arterial pressure monitoring (telemetry)</td></tr><tr><td>Re-gel syringe</td><td>Data Sciences International; Minneapolis, MN</td><td>276-0038-001</td><td>Transmitter reuse (telemetry)</td></tr><tr><td>Disposable pressure transducer</td><td>Transpac; San Clemente, CA</td><td>MI-1224</td><td>Mean arterial pressure monitoring</td></tr><tr><td>Clear-Cuff pressure infuser</td><td>MILA International Inc.; Florence, KY</td><td>2281339</td><td>Mean arterial pressure monitoring</td></tr><tr><td>Vessel cannulation forceps</td><td>Fine Science Tools; Foster City, CA</td><td>00574-11</td><td>Catheter insertion</td></tr><tr><td>Black monofilament nylon 4-0 suture on reverse cutting needle</td><td>McKesson Medical-Surgical; San Francisco, CA</td><td>S661GX</td><td>Secure telemetry transmitter</td></tr><tr><td>Telemetry receiver</td><td>Data Sciences International; Minneapolis, MN</td><td>RPC-1</td><td>Mean arterial pressure monitoring (telemetry)</td></tr><tr><td>LabChart Pro (software), PowerLab (acquisition hardware)</td><td>AD Instruments; Colorado Springs, CO</td><td>ML846, MX2 matrix 2.0 (Compatible with Data Science International telemetry)</td><td>3 options for software/acquisition hardware</td></tr><tr><td>SciWorks (software), DataWave (acquisition hardware)</td><td>DataWave Technologies, Loveland, CO</td><td>N/A</td><td></td></tr><tr><td>Spike 2 (software), Micro1401-3</td><td>Cambridge Electronic Design Ltd., London UK</td><td>1401-3</td><td></td></tr><tr><td>Micro-drill</td><td>Roboz Surgical Instrument Co.; Gaithersburg, MD</td><td>RS-6300</td><td>CNS surgery</td></tr><tr><td>Stereotaxic surgery frame</td><td>Stoelting; Wood Dale, IL</td><td>51600</td><td>CNS surgery</td></tr><tr><td>Microelectrode amplifier with 10X pre-amplifier</td><td>A-M Systems; Sequim, WA</td><td>1800-2</td><td>RSNA recording</td></tr><tr><td>Retractors</td><td>Fine Science Tools; Foster City, CA</td><td>17009-07</td><td>RSNA surgery</td></tr><tr><td>Micro-dissecting tweezers</td><td>Fine Science Tools; Foster City, CA</td><td>11251-10</td><td>RSNA surgery</td></tr><tr><td>Micro-hook</td><td>Fine Science Tools; Foster City, CA</td><td>10064-14</td><td>RSNA surgery</td></tr><tr><td>Mineral oil</td><td>Fisher Scientific; Waltham, MA</td><td>8042-47-5</td><td>RSNA surgery</td></tr><tr><td>Audio monitor</td><td>A-M Systems; Sequim, WA</td><td>3300</td><td>RSNA surgery</td></tr><tr><td>Silica gel</td><td>Wacker, Munchen; Germany</td><td>RT601A-B</td><td>RSNA surgery</td></tr><tr><td>Electrical clips</td><td>Tyco Electronics; Schaffhausen, Switzerland</td><td>EB0283-000</td><td>Grounding or securing perfusion needle</td></tr><tr><td>Bonn scissors, straight/sharp points</td><td>Roboz Surgical Instrument Co; Gaithersburg, MD</td><td>RS-5840</td><td>Perfusion</td></tr><tr><td>Gavage needle</td><td>Harvard Apparatus; Holliston, MA</td><td>75-0286</td><td>Perfusion</td></tr><tr><td>Masterflex perfusion pump</td><td>Cole-Parmer; Vernon Hills, IL</td><td>7524-10</td><td>Perfusion</td></tr><tr><td>Masterflex platinum-cured silicone tubing</td><td>Cole-Parmer; Vernon Hills, IL</td><td>96410-15</td><td>Perfusion</td></tr><tr><td>Formalin (10% buffered solution; 4 L)</td><td>Sigma-Aldrich; St. Louis, MO</td><td>HT501128</td><td>Perfusion</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich; St. Louis, MO</td><td>S0389</td><td>Cryoprotection</td></tr></tbody></table>

quantifying acute changes in renal sympathetic nerve activity in response to central nervous system manipulations in anesthetized rats

肾交感神经活动 (北美放射学会) 和平均动脉压是心血管和自主研究的重要参数;然而, 在测量和分析这些变量的技术上, 有有限的资源指导科学家。本协议描述了麻醉大鼠北美放射学会和平均动脉压力的测量方法。该议定书还包括在中枢神经系统 (CNS) 操作北美放射学会录音期间访问大脑的方法。北美放射学会记录技术与中枢神经系统的药理、optogenetic 或电刺激兼容。当研究人员在非生存实验中测量短期 (最小到 h) 自主性反应时, 这种方法是有用的, 以解剖与中枢神经系统核相关。该方法不打算用于获得北美放射学会在大鼠的慢性 (生存) 记录。北美放射学会的放电, 平均矫正北美放射学会, 平均动脉压力可以量化和分析进一步使用参数统计试验。本文还介绍了获得静脉通路、记录平均动脉压 telemetrically 和脑内固定的方法。

图 1显示了一个戊巴比妥麻醉鼠的样本北美放射学会和 BP 记录。静脉注射肾上腺素被用来诱发平均动脉压力的增加, 并唤起反射和瞬态 sympathoinhibition4,6。为量化北美放射学会, 对未重叠的十年代段的原始北美放射学会进行了矫正和平均;从每个段中减去噪声估计值。
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Watch this Scientific Journal Video about Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats at JoVE.com

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

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

测量中枢神经系统 (CNS) 的交感神经和心血管反应的方法对于推进神经科学是很重要的。该协议的制定, 以帮助科学家测量和量化的急性肾脏交感神经活动 (北美放射学会) 在麻醉大鼠 (不存活) 的变化。

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

quantifying-acute-changes-renal-sympathetic-nerve-activity-response

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

7.7K 次观看.  University of Illinois at Chicago. 测量中枢神经系统 (CNS) 的交感神经和心血管反应的方法对于推进神经科学是很重要的。该协议的制定, 以帮助科学家测量和量化的急性肾脏交感神经活动 (北美放射学会) 在麻醉大鼠 (不存活) 的变化。

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

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

慢性缩窄大鼠坐骨神经疼痛过敏测试

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

科研

医学

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

一个算法的开发与高位脊髓损伤使用遥测装置进行自主神经异常反射的动物的综合研究

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

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

Renal sympathetic nerves contribute significantly to both physiological and pathophysiological phenomena. Evaluating renal sympathetic nerve activity (RSNA) is of great interest in many areas of research such as chronic kidney disease, hypertension, heart failure, diabetes and obesity. Unequivocal assessment of the role of the sympathetic nervous system is thus imperative for proper interpretation of experimental results and understanding of disease processes. RSNA has been traditionally measured in anesthetized rodents, including mice. However, mice usually exhibit very low systemic blood pressure and hemodynamic instability for several hours during anesthesia and surgery. Meaningful interpretation of RSNA is confounded by this non-physiological state, given the intimate relationship between sympathetic nervous tone and cardiovascular status. To address this limitation of traditional approaches, we developed a new method for measuring RSNA in conscious, freely-moving mice. Mice were chronically instrumented with radio-telemeters for continuous monitoring of blood pressure as well as a jugular venous infusion catheter and custom-designed bipolar electrode for direct recording of RSNA. Following a 48-72 hour recovery period, survival rate was 100% and all mice behaved normally. At this time-point, RSNA was successfully recorded in 80% of mice, with viable signals acquired up to 4 and 5 days post-surgery in 70% and 50% of mice, respectively. Physiological blood pressures were recorded in all mice (116&plusmn;2 mmHg; n=10). Recorded RSNA increased with eating and grooming, as well-established in the literature. Furthermore, RSNA was validated by ganglionic blockade and modulation of blood pressure with pharmacological agents. Herein, an effective and manageable method for clear recording of RSNA in conscious, freely-moving mice is described.

Anesthetized mice exhibit non-physiological systemic blood pressure, which precludes meaningful assessment of autonomic tone given the intimate relationship between blood pressure and the autonomic nervous system. Thus, a novel method to simultaneously record renal sympathetic nerve activity and blood pressure with intravenous infusion in conscious mice is outlined.

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 ...

Cavernous Nerve Stimulation and Recording of Intracavernous Pressure in a Rat

The stimulation of the cavernous nerve (CN) and measurement of intracavernous pressure (ICP) have been used extensively to test and evaluate therapies for erectile dysfunction. However, the methods used vary between laboratories, and pitfalls still exist. The goal of this study was to describe a surgical technique that would provide a reliable and reproducible model. By exposing the ischiocavernosus muscle at its point of insertion on the ischial tuberosity, the penile crus could be cannulated with minimal dissection and injury to the structures involved in erectile function. Repeated stimulation of the CN, without the need for lifting and drying, was achieved by using a 125 &#181;m bipolar silver electrode and biocompatible silicon glue to isolate the electrode-nerve complex. This method prevents neuropraxia by reducing stretching and drying the nerve and provides complete isolation of the nerve, negating electrical leakage and preventing stimulation of alternative pathways.

This study describes a simplified surgical procedure and technique for performing cavernous nerve stimulation with the isolation of the nerve-electrode complex using silicone glue and intracavernous pressure measurement.

大鼠海绵状神经刺激与海绵压力记录

The stimulation of the cavernous nerve (CN) and measurement of intracavernous pressure (ICP) have been used extensively to test and evaluate therapies for ...

Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion

The benefits of renal sympathetic denervation (RDN) on blood pressure have been proved in a large number of clinical trials in recent years. However, the regulatory mechanism of RDN on hypertension remains elusive. Thus, it&#39;s essential to establish a simpler RDN model in mice. In this study, osmotic mini pumps filled with Angiotensin II were implanted in 14-week-old C57BL/6 mice. One week after the implantation of the mini-osmotic pump, a modified RDN procedure was performed on bilateral renal arteries of the mice using phenol. Age-sex-matched mice were given saline and served as sham group. Blood pressure was measured at baseline and every week subsequently for 21 days. Then, renal artery, abdominal aorta and heart were collected for histological examination using H&#38;E and Masson staining. In this study, we present a simple, practical, repeatable, and standardized RDN model, which can control hypertension and alleviate cardiac hypertrophy. The technique can denervate peripheral renal sympathetic nerves without renal artery damage. Compared to previous models, the modified RDN facilitates the study of the pathobiology and pathophysiology of hypertension.

Here, we present a protocol for renal sympathetic denervation (RDN) in mice with hypertension induced by Angiotensin II infusion. The procedure is repeatable, convenient and allows to study the regulatory mechanisms of RDN on hypertension and cardiac hypertrophy.

改善肾去神经支配缓解血管紧张素II.诱导的高血压

The benefits of renal sympathetic denervation (RDN) on blood pressure have been proved in a large number of clinical trials in recent years. However, the ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

部分坐骨神经结扎术:慢性神经性疼痛的小鼠模型，用于研究新疗法的抗伤害感受作用

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...

改进大鼠模型中的 FSN 治疗:最大限度地减少不适并最大限度地提高效率

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the mechanisms of action are still not fully understood. During FSN treatment, the FSN needle is inserted and retained in the subcutaneous tissues for a long duration with a swaying movement. However, challenges arise from maintaining a posture while manipulating FSN in animal models (e.g., rats) for researchers. Uncomfortable treatment can lead to fear and resistance to FSN needles, increasing the risk of injury and may even affect research data. Anesthesia may also affect the study results too. Hence, there is a need for strategies in FSN therapy on animals that minimize injury during the intervention. This study employs a chronic constriction injury model in Sprague-Dawley rats to induce neuropathic pain. This model replicates the pain induced by nerve injury observed in humans through surgical constriction of a peripheral nerve, mimicking the compression or entrapment seen in conditions such as nerve compression syndromes and peripheral neuropathies. We introduce an appropriate manipulation for easily inserting an FSN needle into the subcutaneous layer of the animal&#39;s body, including needle insertion and direction, needle retention, and swaying movement. Minimizing the rat&#39;s discomfort prevents the rat from being tense, which causes the muscle to contract and hinder the entry of the needle and improves the study efficiency.

作者聚焦:揭示 FSN 治疗的治疗效果 – 桥接神经性疼痛的研究和临床应用

We present a protocol for using Fu's subcutaneous needling in a chronic constriction injury model to induce sciatic nerve pain in rats.

傅氏皮下针刺治疗坐骨神经疼痛的疗效:慢性缩窄损伤大鼠模型中的行为和电生理变化

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the ...

Sympathetic Denervation of Renal Arteries in an Angiotensin II-Induced Hypertension Mouse Model

Source: Wang, M., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/63719">Improved Renal Denervation Mitigated Hypertension Induced by Angiotensin II Infusion.</a> J. Vis. Exp. (2022)
This video demonstrates a procedure for renal artery sympathetic denervation in an angiotensin II-induced hypertensive mouse model, using phenol to disrupt sympathetic nerve function and reduce blood pressure.

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

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慢性缩窄大鼠坐骨神经疼痛过敏测试

一个算法的开发与高位脊髓损伤使用遥测装置进行自主神经异常反射的动物的综合研究

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。

大鼠海绵状神经刺激与海绵压力记录

改善肾去神经支配缓解血管紧张素II.诱导的高血压

部分坐骨神经结扎术:慢性神经性疼痛的小鼠模型，用于研究新疗法的抗伤害感受作用

傅氏皮下针刺治疗坐骨神经疼痛的疗效:慢性缩窄损伤大鼠模型中的行为和电生理变化

傅氏皮下针刺治疗坐骨神经疼痛的疗效:慢性缩窄损伤大鼠模型中的行为和电生理变化

傅氏皮下针刺治疗坐骨神经疼痛的疗效:慢性缩窄损伤大鼠模型中的行为和电生理变化

Sympathetic Denervation of Renal Arteries in an Angiotensin II-Induced Hypertension Mouse Model