作者衷心感谢凯蒂. 芬克和林恩 Dondlinger 在准备这份手稿方面的宝贵帮助。
补助金支持: NIH #R21-OD018309;#R56 HL065289;还有 #R01 HL128242

威斯康星医学院动物护理和使用委员会 (IACUC) 批准了本文所述的所有协议, 所有程序都符合国家卫生研究院 (NIH) 实验室动物福利办公室 (OLAW)条例.
1. 溶液和容器室的制备
<ol><li>在进行一系列实验之前, 准备 2 l 20x 浓盐溶液, 由278克/L 氯化钠组成;14克/升氯化钾;11.52 克/升 MgSO4.7H2O;和9.4 克/L CaCl2.2H2O。还准备2升的20x 集中缓冲库存, 由80.8 克/升 NaHCO3和0.4 克/l EDTA 组成, 2 l 的20x 集中的 Ca2 +的股票解决方案由281.6 克/l 氯化钠组成;14克/升氯化钾, 11.52 克/升 MgSO4.7H2O。 注: 20X 库存解决方案可存储在冰箱中, 直到使用。</li>
	<li>在实验的当天, 从20x 集中库存溶液中制备 2 l 的生理盐溶液 (PSS), 如下所示: 将100毫升的20x 盐库存添加到1800毫升的去离子水中, 在一个2升锥形烧瓶或一个机动搅拌盘上的烧杯中。添加100毫升的20x 缓冲库存, 同时不断均衡的解决方案与气体混合物包含 21% O2, 5% CO2, 平衡 N2, 并搅拌它与磁性搅拌棒。在监测 pH 值时, 慢慢地添加 0.28 g NaH2PO4 ;根据需要对 pH 值7.4 进行调整, 从巴斯德吸管中加入6氮 HCl 或 6.5 n 氢氧化钠溶液。在 pss 准备好并调整 pH 值后, 将1.98 克葡萄糖添加到 pss 中。 注意: 在监测 PSS ph 值时, 必须缓慢添加 NaH2po4 , 因为在碱性溶液 (ph > 7.4) 中加入 NaH2po4可以形成磷酸钙沉淀, 如在容器底部有一个混浊的溶液或白色沉淀的外观。 注: PSS 的最终组成为119毫米/升氯化钠;4.7 毫米/升氯化钾;1.17 毫米/升 Mg2, 因此4;1.6 mM/升 CaCl2;1.18 mM/升 NaH2PO4;24 mM/升 NaHCO3;0.03 毫米/升 EDTA;和5.5 毫米/升葡萄糖。虽然 PSS 的组成可以在实验室之间有所不同, 但这一食谱非常适合于维持血管张力, 内皮功能, 和对血管活性物质的反应, 在孤立的阻力动脉。</li>
	<li>为了确定最大的直径, 并通过产生最大的血管扩张来评估动脉内的活动音调, 通过将 20X ca 的25毫升2 +的盐量添加到450毫升的去离子水中, 准备500毫升的 ca2 +释放 PSS。, 后跟25毫升的20x 缓冲储存在一个锥形烧瓶或烧杯类似于步骤1.2 以上。在监控和调整解决方案的 pH 值时, 将0.07 克 NaH2PO4添加到解决方案中。在实验结束时, 将 ca2个无的 pss 添加到 pss 库和容器室, 以避免耗尽在正常 pss 中可能影响容器响应的胞内 ca2 +存储。由于在实验结束时添加了 Ca2 +-免费解决方案, 以产生最大程度的动脉松弛, 因此不需要向 PSS 添加葡萄糖。 注: 当完成后, Ca2 +的最终组成为120.6 毫米/升氯化钠;4.7 毫米/升氯化钾;1.17 毫米/升 Mg2, 因此4;1.18 mM/升 NaH2PO4;24 mM/升 NaHCO3;0 mM/升 CaCl2;和0.03 毫米/升 EDTA。 注: 对于许多需要最大动脉扩张的研究, 除了从 PSS 中移除 Ca2 +之外, 还可以在解决方案中添加一个钙进入阻滞剂, 如维拉帕米 (1 µM) 和/或一氧化氮供体 (如硝普钠 (10 µM))。</li>
	<li>保持 PO2、不正常情况下的2和 pss 的 pH 值, 持续均衡在标准的器官浴中流入血管室的 pss, 用于研究孤立的主动脉环、肠道平滑肌或其他组织 (图 1)。使用合成氟聚合物的四氟乙烯油管连接气罐到器官浴, 因为这种类型的油管是气体不透水, 与许多其他形式的油管,例如, 乳胶。</li>
	<li>在容器室中放置一个连接到平衡气体混合物的小型空气石, 以帮助维持 PSS 气体成分。 注: 船只对 PO2变化的反应可以通过均衡容器室中的 PSS 和腔液的气体混合物进行测试, 其中含有不同百分比的 O2,例如, 21% o2, 10% o2, 5% o2, 和 0% O2, 具有 5% CO2和平衡 N233,34,35。对于较厚的动脉, 如果将氧气扩散到血管壁的中心可能是一个限制, 更高的氧气百分比,例如, 95% O2可以使用。</li>
	<li>密切监视容器内的温度, 因为单个腔室的传热特性可能会有所不同。 注: 用于研究空心阻力动脉的许多商用导管室利用蠕动泵从气平衡油藏输送含氧 PSS, 并提供非常精确的浴温和氧的控制PSS。</li>
	<li>将 pss 放在一个大 (2 升) 马里奥特瓶中, 用塞子和中央玻璃管作为蓄水池, 将 pss 源源不断地送到加热和气体均衡的机构浴中, 使 pss 流入容器室 (图 1A)。</li>
	<li>将中央玻璃管的开口放在马里奥特瓶中, 与 pss 在风琴浴中的顶部一样, 保持恒定的静水压头, 以便将 pss 送到器官浴中。使用连接到 J 型玻璃或塑料管的聚乙烯导管将 PSS 从马里奥特瓶中送到器官浴中。</li>
	<li>对于腔灌注 (图 1A), 使用聚乙烯导管将流入的吸管连接到一个由60毫升塑料注射器组成的 PSS 油藏, 它的位置可维持所需的流入压力 (通常为研究大鼠大脑的80柱。动脉), 测量用一个压力传感器连接到系统通过一个塞。</li>
	<li>将流出的吸管连接到聚乙烯管, 使 PSS 能够通过容器来响应压力梯度, 并将流出线连接到类似流入水库的水库。使用类似的塞和压力传感器连接来测量流出压力。 注: 下面2节描述了设置壁压力和通过容器控制流量的程序。</li>
	<li>在实验结束时, 用蒸馏水彻底冲洗房间、输送管线和储水池系统。在频繁的时间间隔内, 更换油管和输送线, 清洁或更换系统中的三通, 并周期性地将任何玻璃 PSS 储油层置于酸洗中, 以防止细菌和其他引起污染的微生物生长,影响船只的反应性。</li>
</ol>2. 空心动脉的制备
<ol><li>麻醉一大5% 异氟醚的大鼠, 用 1.5-2.5% 的医用级氧36维持麻醉。另外, 管理肌肉注射氯胺酮 (75.0 毫克/千克), 乙酰 (2.5 毫克/千克), 和嗪 (10.0 毫克/千克);腹腔注射戊巴比妥 (50-60 毫克/千克);或任何其他认可的麻醉方法, 取决于协议和/或调查者的喜好。</li>
	<li>斩首深麻醉下的大鼠, 取出脑血管进行脑阻力研究。</li>
	<li>在切除大脑后, 仔细地隔离中动脉 (MCA) (或其他感兴趣的动脉,如, 基底动脉或后脑动脉)37,38。为了隔离 MCAs, 将大脑仰卧在一个充满冰冷 PSS 的玻璃培养皿中 (图 2)。</li>
	<li>使用 Vannas 剪刀和一个 #5 精尖钳, 从大脑中切除 MCA。 使用镊子从 MCA 中清除任何残留的脑组织, 并将动脉转移到装有 PSS 的温度控制容器室, 如前所述。33,34 .</li>
	<li>将动脉转移到血管室, 用 ACA 或后向交流动脉段轻轻握住切除血管, 并小心地将其放入会议厅。 注: 除 MCA 外, 中空血管系统适用于各种小血管的制备, 包括骨骼肌阻力动脉33、39、40、肠系膜阻力动脉38,41,42, 和大 (一阶) 的睾肌的动脉43, 以及人冠状动脉和人动脉从皮下脂肪组织的臀部活检中获得44,45, 46,47。</li>
	<li>将动脉连接到流入的微, 把它拉向吸管底座, 直到尖端进入 MCA 的流明。在流入的吸管上固定动脉, 通过结扎从10-0 条缝合线 (图 1B) 的单链纤维制成的环。通过在容器周围拧紧第二个缝合环 (图 1B), 将 MCA 的另一端固定到流出吸管上。 注: 将缝合环放在 micropipettes 上, 然后将其放置在靠近最后的附着点处, 使它们容易滑过动脉, 并在血管就位时迅速获得保护, 从而使动脉滑出管。 注: Micropipettes 是由硼硅酸盐玻璃毛细管 (2 毫米外径; 1 毫米内径; 10 厘米长) 用立式微拉拔器制成。在连接动脉之前, 尽量匹配 micropipettes 的尖端直径, 以防止灌注系统中流入和流出阻力的不匹配。</li>
	<li>在动脉被安全地绑在 micropipettes 上后, 使用与流入吸管支架相连的千分尺将动脉伸展至其原位长度。</li>
	<li>从10-0 条缝合线上的单绞线系住所有的侧支, 以便在动脉中保持恒定的压力。</li>
	<li>通过确保在临时关闭流入吸管后的腔内 (壁) 压力保持恒定, 验证是否存在泄漏。如果压力下降, 在船上系上任何树枝或检查洞。恢复灌注后, 验证壁压力保持恒定。 注: 隔离阻力动脉插管需要人工灵巧和练习。要注意的主要预防措施是避免破坏管, 并确保动脉不会滑出管。在整个手术过程中, 对孤立的动脉进行轻柔的处理是很重要的, 因为血管的创伤会损害血管平滑肌的内皮和/或干扰正常功能。</li>
	<li>使用视频显微镜设置 (图 1A) 测量动脉内径, 它由连接在解剖显微镜上的摄像机组成, 并连接到视频千分尺和电视显示器 (数字 1B, 1C)。这样, 观察者就可以通过在动脉的内壁上放置可移动的参考线, 以及如果需要, 在动脉的外壁上, 测量船只的直径, 以便测量容器壁厚。 注: 一些视频千分尺提供船舶尺寸的自动跟踪。 注: 在实验之间用显微镜级千分尺和流入流出压力传感器 (0 柱、50柱、100柱、150柱和200柱) 校准视频千分尺, 以确保准确测量容器直径和腔内压力。 注: 大鼠 MCA 实验的标准控制壁压力为80柱。较高和较低压力水平的校准确保了研究壁压力和最大扩张动脉内被动压力-直径曲线的肌源反应的准确性。</li>
	<li>调整流入和流出油藏的高度, 以保持所需的壁压力恒定水平。以少量 (< 5 柱) 来提高流入水库的数量, 并以同样的量来降低流出油藏, 维持平均壁压力, 并在容器流明6中产生灌注流。 注: 提高流入油藏和降低流出库的等量量, 保持了动脉内相同的平均壁扩张压力, 但产生了静水压力梯度, 导致血管内的流动和剪应力, 使研究人员对不同实验组的腔内剪应力变化的内皮依赖性反应进行评估48。</li>
	<li>为了评估肌源性反应和血管舒张刺激的反应, 确保动脉在实验前表现出适当的活动音水平 (大约 40%)。丢弃任何缺乏活动音的动脉, 除了血管,例如, 小肠系膜动脉通常不表现出活跃的休息音。 注: 对于通常不具有自发色调的血管, 使用收缩激动剂, 如去甲肾上腺素或肾上腺素, 预先收缩动脉的当量。选择用于预收缩动脉的激动剂剂量的一个好方法是利用 EC50剂量的收缩剂,如, 去甲肾上腺素41,42。然而, 在静止状态下, 药物收缩剂不应用于表现自发活动音的动脉。</li>
	<li>通过测量血管直径在增加乙酰胆碱浓度 (10-10 M–10-5 M) 到容器室的过程中, 测试空心阻力动脉的内皮依赖性反应。在增加浓度 (10-10 M–10-5 M) 的情况下测量血管平滑肌的内皮独立一氧化氮敏感性容器室。 注意: 对收缩剂和其他血管扩张性激动药的敏感性可以通过在容器腔中加入增加的浓度来测试类似的方式。 注: 除血管活性物质外, 各种药物、抑制剂和其他药理制剂均可添加到组织浴和/或腔液。PO2 (以及腔的2和 pH 值) 的变化可以通过腔液的分离平衡和与标定的气体混合物的空气石进行选择性地对动脉的内皮侧或额外的外侧进行选择管理。不同于在组织浴中用于平衡 PSS 的混合物33,34,49。通过关闭流出管并提高 (或降低) 连接到流入吸管的 PSS 油藏的高度 (49,50,51 , 可以研究对壁压力变化的肌反应。增加或减少腔内压力。</li>
	<li>为了测试内皮在调节血管对特定刺激的反应中的作用, 去除血管内皮, 并在内皮存在或缺失的情况下比较血管的反应。为了去除血管内皮, 小心地从流出的吸管中松开动脉, 用空气丸 (0.5-1.0 毫升) 慢慢灌注动脉腔。经空气灌流后, 恢复 PSS 灌注, 清除细胞碎片, 然后再将容器重新绑到流出液管43。 注意: 在内皮剥脱术后, 重要的是要通过检测血管反应 (通常为乙酰胆碱) 来验证内皮细胞, 以产生血管内皮依赖性血管舒张。然而, 在某些病理条件下, 内皮依赖性收缩被释放, 在这种情况下, 它是重要的, 以验证在血管内皮切除后的大蟒反应消除。</li>
	<li>在实验结束时, 通过在液和 superfusate 上添加 Ca2 +无 PSS, 确定动脉的最大直径。计算活动休眠音 (%) 为 (d最大-drest)/d最大) x 100, 其中 dmax是存在于 Ca2 +无解决方案中的最大直径, drest是静止控制直径。 注: 不同实验组的最大松弛动脉直径测量在比较活动休眠音、结构重塑 (即、壁厚和流明直径的变化) 和被动机械性能方面有价值(应力-应变关系计算从被动直径在不同水平的壁压力)。</li>
</ol>3. 对脑血流反应的评价
<ol><li>用5% 异氟醚麻醉动物, 在立体定向仪中保护鼠,9,10。</li>
	<li>在监测呼吸频率, 结束潮汐 CO2, 麻醉深度和脚趾掐36的情况下, 保持动物在恒定的麻醉状态。</li>
	<li>小心薄颅骨半透明使用低速牙科钻和矿物油提供光耦合10。注意避免产生过多的热量, 避免穿透骨头。 注意: 颅骨变薄允许激光照射到底层组织, 并反射回探针以测量多普勒位移, 其大小由移动粒子 (即, 红细胞) 的数量决定。他们的速度。</li>
	<li>在微的地方保护自卫探针, 并将其直接放置在头骨的稀疏区域。在实验中, 这是非常重要的, 以防止运动的自卫的探针或准备本身, 因为防御系统的设计, 以测量流量在一个限制区域的组织和对运动文物非常敏感。 注意: 探针的任何移动从它的最初的位置将提供估计血液流动在组织的不同的区域, 排除比较。虽然非没有提供绝对的流量值, 也不适合在实验对象之间进行比较, 但它是一个很好的方法来评估组织灌注的变化, 以响应个别学科的试验性干预;从控制值可以平均和相对变化的自卫队信号从控制在其他实验组的变化。 注意: 在不同实验组的整个血管床层的血流量的调节因素中, "自卫队" 是一个方便的方法8,9,10。组织灌注的评价提供了一个切实可行的方法, 吸收从孤立的船只研究的知识, 以整个床的角度。除非在阻力动脉和微循环之间的血管控制机制上存在区域差异, 否则, 用自卫法获得的测量结果可以很好地表明组织的血流量控制, 这通常与获得空心动脉的准备。</li>
</ol>4. GS1 凝集素对骨骼肌微血管密度的评价
<ol><li>用标准精细的手术剪刀切开阴囊, 然后用 #5 钳抓住肌肉, 从雄性大鼠身上取出睾肌肉。 注意: 瘦肌肉 (例如, 睾肌, 以及在雄性和雌性大鼠中发现的伸肌腱长长和胫骨前肌) 都适合用作凝集素研究的整体支架, 尽管组织学切片可用于较厚的组织。</li>
	<li>使用单切口从睾丸中取出睾肌。将它放在冰冷的 PSS 中, 并将其固定在一个培养皿中, 在底部内表面有硅胶弹性体衬里。用 #5 钳将结缔组织轻轻地梳理掉。</li>
	<li>用2毫升缓冲的 PSS 冲洗肌肉样本, 然后将它们浸泡在罗丹明标记的 GS1 凝集素 (20 µg/毫升 pss) 中, 在12井细胞培养板中50分钟, 用铝箔包裹的2毫升/井, 以排除光。</li>
	<li>从凝集素溶液中取出组织, 在 PSS 中冲洗三次, 用5分钟的 "洗涤" 孵化在摇杆上, 然后将其贴在显微镜幻灯片上。确保在黑暗中孵育组织, 并在黑暗中存储幻灯片, 以防止失去荧光。 注意: 如果不能立即使用幻灯片, 可以将它们保存在冰箱中, 而不会丢失荧光。对于长时间存储, 可以将幻灯片保存在冰箱中以防止损坏11。</li>
	<li>通过在图像52上叠加计算机生成的网格线, 或者在用于查看幻灯片的监视器上叠加一个清晰的网格叠加, 来计算微血管密度。 注: GS1 凝集素方法已被用来证明: 盐诱导微血管稀疏53, 预防盐诱导血管紧张肽 II. 抑制盐喂养动物微血管密度的保护作用17 ,53,54;NRF2 介导低剂量血管紧张素 II. 在预防盐喂养大鼠微血管稀疏中的保护作用17;并评价血管紧张素 II 在维持盐大鼠慢性肌肉刺激血管生成反应中的作用54,55。GS1 凝集素技术的一个优势是, 它可以用来评估微血管密度在相同的动物用于研究空心阻力动脉或自卫。</li>
</ol>

作者没有相互竞争的金融利益。

正如导言中所指出的, 本文介绍了电视显微镜和孤立阻力动脉的方法, 以评估血管功能不仅在标准大鼠模型 (在视频中使用), 而且在高度专门化的基因工程大鼠菌株, 这表明了小说和强大的洞察力, 可以得到利用这些方法。利用这些强大的技术来评估小阻力动脉的主动音和被动力学特性, 可以提供关于多种血管控制机制的重要信息, 包括内皮依赖性调节在正常和病理生理条件下, 血管平滑肌功能与血管...

动脉血管功能的最早研究利用导管动脉, 在许多情况下是主动脉。通过将动脉环段附着在组织浴中的力传感器上, 对大动脉的力产生进行了研究;在主动脉的情况下, 通过切割血管的螺旋条, 使平滑肌纤维在附着点和力传感器之间的纵向方向上定向, 以提供由收缩产生的力的最佳估计沿其纵轴的平滑肌肉。切割脉螺旋条的标准技术是在容器的流明处放置一根玻璃棒, 在容器壁上以所需的角度进行切割, 并在容器壁的外露边缘的一端保持, 因为切口被延伸以产生整个螺旋带的船只。在这一点上, 血管的内皮侧通常被涂抹, 以去除碎片之前, 连接到力传感器的船只带和淹没准备在一个氧和温度控制组织浴。最终, 这种方法导致了在生理学史上最著名和最重要的发现之一, Furchgott 和 Zawadski1, 即内皮衍生舒张因子 (EDRF) 的作用, 后来被确定为一氧化氮, 在调节血管功能。导致这一发现的关键事件是, 在这种情况下, 研究者通过避免动脉与外表面的内皮侧接触来维持完整的内皮, 并注意到主动脉带没有表现出预期收缩到乙酰胆碱, 但在反应中松弛。根据这一观察, 调查人员研制了一种 "三明治" 制剂, 在其中将主动脉段与一个完整的内皮细胞 (但不能产生收缩力) 连接到主动脉的标准螺旋带, 并转化为收缩成一个放松。
今天广泛使用的两个主要进展是在小阻力动脉中测量主动收缩力的准备工作的发展2,3 (如肠道肠系膜上的那些)3) 和空心阻力动脉准备4,5,6。在最早的一份报告中, Mulvany 和 Halpern3描述了使用金属丝 myograph 的制备方法, 研究自发性高血压大鼠 (SHR) 肠肠系膜上孤立阻力动脉的主动收缩力, 并正常 WKY 控制。在导线 myograph 系统的发展之后, 空心抵抗动脉准备被开发了允许研究更接近在体内情况4,5,6。 虽然这两种方法都提供了有价值的结果, 但空心动脉的制备具有更有效地保持动脉内固有活动张力的优点;并允许研究人员对壁压力和血管反应对流速和内皮剪切力变化的影响进行活性的肌源反应 (参见 Halpern 和凯利6的回顾)。
本论文的主要目的是描述如何使用孤立的, 空心阻力动脉的视频显微术的时间, 以获得准确的信息, 有关的机制, 调节主动语气在这些关键血管, 独立于神经、体液或实质细胞的影响。这一基本信息, 使用一个标准的大鼠模型和我们的研究新的基因工程大鼠菌株的例子, 将提供给读者一个关于血管功能的见解类型的想法, 可以获得与电视显微镜方法, 可用于研究涉及任何控制和实验组的研究者的选择, 包括强大的新的实验大鼠模型的选择性近亲繁殖和新发展的遗传工程技术。
由于电视显微镜方法的精确度, 测量空心动脉的直径变化可为血管内皮依赖性和内皮独立机制提供极有价值的信息。放松, 以及在高血压、高盐饮食和其他实验干预的血管控制机制中发生的重要 (有时是意想不到的) 改变。此外, 测量的压力-直径关系的孤立和空心阻力的动脉, 最大限度地放宽了治疗的 Ca2 +免费的解决方案或药理血管扩张药物, 使调查员评估血管重塑和计算被动应力-应变关系的动脉结构变化7可以提供重要的洞察力改变动脉的被动机械特性, 从而影响动脉功能独立于 (或除了) 主动控制机制的变化。还必须指出的是, 从隔离的阻力动脉的研究中获得的信息可以补充利用获得的信息, 这是一种评估整个动物级别组织灌注的实用方法8,9 ,10, 以及通过使用荧光标记的 GS1 凝集素来评估微血管密度所获得的信息, 这特别结合了小动脉和毛细血管基底膜的糖蛋白基11,12. 后一种方法提供了一个高度准确的微血管密度的估计, 不受传统的困难, 在估计微血管密度通过计数血管的在体内, 例如失踪非灌注由于动脉主动闭合而导致血流停止的血管。如果一起使用, 这些方法可以提供重要的洞察力相关的功能改变的孤立阻力动脉的变化, 组织灌注在微循环水平;在本手稿中还将提供一些使用这些有价值的方法与空心动脉技术结合的例子。
本文主要研究利用视频显微镜技术对 outbred 大大鼠动脉血管的变化进行评价。然而, 重要的是要注意, 这些技术已证明是非常宝贵的, 以阐明表型变化的高度专业化的基因工程大鼠选育或基因编辑使用的技术。在这篇手稿中, 我们提供的例子, 视频显微技术如何提供了重要信息的血管功能, 在许多有价值的大鼠模型, 包括达尔盐敏感 (SS) 大鼠-一个近交系大鼠菌株, 是最广泛的用实验模型研究了盐敏 hypertenson 的机理18、19、20、21、22、23;和 consomic 大鼠通过选择性选育 SS 大鼠与盐不敏感的棕色挪威 (BN) 大鼠品系。在 consomic 鼠板中, 来自棕挪威鼠的每一个染色体都被单独渗入到达尔 SS24,25,26遗传背景。使用 consomic 鼠板提供了宝贵的线索, 关于特定的染色体, 有助于盐敏感性血压和其他表型, 包括血管反应性24,25,26 ,27,28。
利用 ss 大鼠和携带个别 BN 染色体的 consomic 大鼠的选择性育种策略, 也使同类菌株的小片段渗入到达尔 ss 遗传背景22,29。这些可以提供非常有价值的输入对特定的基因或染色体的狭窄区域, 可以影响关键的生理变量, 如血压, 肾脏损害, 和血管活性22,29。大鼠基因工具箱的另一个强大的补充是利用先进的基因编辑技术, 包括 ZFNs, 转录激活剂类似效应核酸 (TALENS), 最近 CRISPR-Cas913 ,14,15,16,17。这些强大的技术的问世, 使基因被淘汰的老鼠是一个非常重要的发展, 因为基因剔除研究迄今已使用 (并继续使用) 小鼠几乎完全。本论文的另一项实验研究表明, 空心动脉技术和视频显微术对无主抗氧化剂和细胞保护转录的击倒大鼠的生理控制机制有价值因子, 核因子 (红-衍生 2) 样-2 (NRF2)30,31, 这是利用塔伦技术在大遗传背景17中开发的。在这些实验中,体外视频显微镜技术用于提供 NRF2 基因丢失的功能验证, 并在 NRF2-mediated 抗氧化剂直接上调的基础上测试一种潜在的有价值的治疗方法。防御.NRF-2 在对抗血管氧化应激方面具有重要的治疗作用, 因为临床试验的结果令人失望, 例如维生素 C 和 E32直接管理抗氧化剂。

<table>
	<tbody>
		<tr>
			<td>SS Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS/JHsd/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>SS.5BN Consomic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS-Chr 5BN/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>SS.13BN Consomic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS-Chr 13BN/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Ren1-BN Congenic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS.BN-(D13hmgc41-D13)hmgc23/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Ren1-SSA Congenic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS.BN-(D13rat77-D13rat105/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Ren1-SSB Congenic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS.BN-(D13rat124-D13rat101/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Nrf2(-/-) Knockout Rat and Wild Type Littermates</td>
			<td>Medical College of Wisconsin</td>
			<td>SD-Nfe212em1Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Low Salt Rat Chow (0.4% NaCl)-AIN-76A</td>
			<td>Dyets, Inc.</td>
			<td>113755</td>
			<td></td>
		</tr>
		<tr>
			<td>High Salt Rat Chow (4% NaCl)-AIN-76A</td>
			<td>Dyets, Inc.</td>
			<td>113756</td>
			<td></td>
		</tr>
		<tr>
			<td>Colorado Video Caliper</td>
			<td>Colorado Video, Inc.</td>
			<td>Model 308</td>
			<td></td>
		</tr>
		<tr>
			<td>Video Camera</td>
			<td>Hitachi</td>
			<td>KPM1AN</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus Life Science</td>
			<td>CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td>Television Monitor</td>
			<td>Panasonic</td>
			<td>WVBM1410</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Transducers</td>
			<td>Stoelting</td>
			<td>56360</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Pressure Display Unit</td>
			<td>Stoelting</td>
			<td>50115</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannulated Artery Chamber</td>
			<td>Living Systems Instrumentation</td>
			<td>CH-1</td>
			<td>Single vessel chamber for general use</td>
		</tr>
		<tr>
			<td>Temperature Controller for Single Chamber</td>
			<td>Living Systems Instrumentation</td>
			<td>TC-09S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Dispersion Tube, Miniature,Straight</td>
			<td>Living Systems Instrumentation</td>
			<td>GD-MS</td>
			<td>Provides aeration in the vessel bath</td>
		</tr>
		<tr>
			<td>Gas Exchange Oxygenator, Miniature</td>
			<td>Living Systems Instrumentation</td>
			<td>OX</td>
			<td>Allows gas exchange with perfusate</td>
		</tr>
		<tr>
			<td>Laser-Doppler Flowmeter</td>
			<td>Perimed</td>
			<td>PeriFlux 5000 LDPM</td>
			<td></td>
		</tr>
		<tr>
			<td>GS1 Lectin</td>
			<td>Vector Labs</td>
			<td>RL-1102</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillary Tubes for Micropipettes</td>
			<td>Fredrich Haer Co.</td>
			<td>27-33-1</td>
			<td>2 mm ODX1 mm ID</td>
		</tr>
		<tr>
			<td>Verticle Pipette Puller</td>
			<td>David Kopf Instruments</td>
			<td>Model 700C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon suture material (10/0)-3 PLY</td>
			<td>Ashaway Line and Twine Manufacturing Co.</td>
			<td>114-ANM-10</td>
			<td>Single strands of 3 ply nylon suture teased out for use on vessels</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps-Inox</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors</td>
			<td>Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Protandim</td>
			<td>Protandim</td>
			<td></td>
			<td>NRF2 Inducer: Contact Dr. Joe McCord (JOE.MCCORD@UCDENVER.EDU)</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Bioreagents</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher Chemical</td>
			<td>S233-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose (d-glucose) anhydrous</td>
			<td>Fisher Chemical</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (MgSO4-7H2O)</td>
			<td>Sigma Aldrich</td>
			<td>M1880-500 G</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2-2 H2O)</td>
			<td>Sigma</td>
			<td>C5080-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate-Monobasic (NaH2PO4)</td>
			<td>Sigma</td>
			<td>S0751-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Chemical</td>
			<td>P217-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td>Sigma</td>
			<td>ED255-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of vascular control mechanisms utilizing video microscopy of isolated resistance arteries of rats

该协议描述了使用在体外电视显微镜来评估孤立的脑阻力动脉 (和其他血管) 的血管功能, 并描述了使用激光多普勒血组织灌注的评价技术 () 和微血管密度利用荧光标记的加纳单叶(GS1) 凝集素。目前的方法研究孤立阻力动脉在壁压力遇到在体内和在缺乏实质细胞的影响提供了一个关键的联系之间的体内研究和信息获得的分子简化的方法, 提供有限的洞察力的整体反应在整个动物的水平。荧光标记的 GS1 凝集素有选择地识别动脉和毛细血管的方法, 提供了切实可行的解决方案, 使调查人员能够扩大从孤立阻力动脉的研究中获得的知识。本文介绍了这些技术在大鼠血管生理学和病理学基础知识中的应用, 作为一个一般的实验模型, 并在各种专门的基因工程 "设计" 大鼠菌株, 可以提供重要的洞察力特定基因对重要血管表型的影响。利用这些有价值的实验方法, 在大鼠的选育策略和新技术的培育下, 建立基因敲除模型, 将扩大在挖空小鼠模型中开发的科学前提的严谨性, 并将这一知识扩展到一个更相关的动物模型中, 因为它的尺寸较大, 具有很好的生理背景和生理研究的适用性。

体外空心阻力动脉显微镜允许研究影响小阻力动脉 (和大动脉) 在正常的体内壁压力和缺乏实质细胞的主动音的因素影响.除了评估在正常 pss 中对壁压力升高的各种血管扩张剂和收缩刺激和肌源反应的反应性外, 还可以在液和 superfusate 中添加 Ca2 +-免费的 pss。试验确定了最大容器直径和壁厚。后者的测量是非常有价值的评估血管重塑,即, 改变的?...

Watch this Scientific Journal Video about Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats at JoVE.com

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

这篇手稿描述了体外大鼠脑阻力动脉血管功能的视频显微术。该手稿还描述了用激光多普勒血荧光标记凝集素和组织灌注评价微血管密度的技术。

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

evaluation-vascular-control-mechanisms-utilizing-video-microscopy

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

9.2K 次观看.  Marquette University. 这篇手稿描述了体外大鼠脑阻力动脉血管功能的视频显微术。该手稿还描述了用激光多普勒血荧光标记凝集素和组织灌注评价微血管密度的技术。

视频: Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2. 
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.

Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.

在单绒毛膜胎盘血管吻合用有色染料的精确和简单的评价

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

科研

医学

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

培养一个精密的显微外科技术在大鼠颈总动脉，颈外静脉移植:介入

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

评估肌反应及血管活性阻力肠系膜动脉使用压力肌动描记

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual&#39;s use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual&#39;s postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person&#x2019;s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

视网膜血流小鼠活体视频显微镜测量

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

实施<em&gt;体外</em&gt;耐药性检测:最大化的潜力，揭开临床有关的耐药机制

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of endothelial function can be achieved by employing the flow-mediated dilation technique, typically performed in the brachial or superficial femoral artery. Briefly, a blood pressure cuff placed distal to an ultrasound probe is inflated to a suprasystolic pressure, which results in limb ischemia. After 5 min of occlusion the cuff is deflated, resulting in reactive hyperemia and increases in wall shear rate that signal vasodilatory molecules to be released from the endothelium eliciting vasodilation. Despite the thousands of studies performing flow-mediated dilation in humans, surprisingly, no studies have performed this technique non-invasively in living rats. Considering the recent shift in focus to translational research, the establishment of guidelines for non-invasive measurement of flow-mediated dilation in rats and other rodents would be extremely valuable. In the following article, a protocol is presented for the non-invasive measurement of flow-mediated dilation in brachial and superficial femoral arteries of rats, as those sites are most commonly measured in humans.

Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats.

肱血流介导的扩张和大鼠股浅动脉超声评估

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

套筒技术在主动脉移植小鼠模型中的应用-教学视频

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

阻力动脉的细胞培养模型

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

利用隔离的完美大鼠小肠进行肠道激素分泌的机制

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...