Name: right ventricular systolic pressure measurements in combination with harvest of lung and immune tissue samples in mice
Uploaded: 2013-01-16T12:00:00.000+00:00
Duration: 10 min 20 s
Description: Watch this Scientific Journal Video about Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice at JoVE.com

这项工作是由国家机构的健康1R21HL092370-01（GG），1R01 HL095764-01（GG）; R01HL082694（JW），美国心脏协会的创始人联属公司（0855943D，GG），石溪世界 - 赫伯特基金，纽约（SHP）。

1。准备<ol><li>准备以下的解决方案和管（ 表1）如下: <ol><li> Hanks溶液，无钙，镁或指示器，与青霉素（100单位/毫升）/链霉素（100毫克/毫升）。 </li><li>磷酸盐缓冲盐水（PBS），1个，无钙，无镁。 </li><li>乙醇，70％，500毫升。 </li><li>与PBS缓冲甲醛，7％至10％，使500毫升。 </li><li>麻醉科的解决方案: <ol><li>圣阿韦坦。小心地加入2 - 甲基-2 - 丁醇的5毫升至5克的2,2,2 - 三溴乙醇。一旦溶解后，在室温下在黑暗中保持原液。以0.25毫升原液和稀释1xPBS在玻璃底瓶用10ml。包裹铝箔，地点的瓶在37℃下，直至溶解，然后等分试样分成5毫升的聚丙烯管中，并保存在冰箱中。温暖的一份日上午的实验。 </li><li>巴比妥类药物。与PBS稀释原液2.6％巴比妥类溶胶ution。将聚丙烯管中的3-4毫升的等分试样。 </li></ol></li></ol></li><li>组织三个工作区（ 图1）。 </li><li><ol><li> A区:安排以下项目:的的学习形式记录研究的识别号码，日期，体重，重量右心，左心和隔垫;精密刻度（0-50克精度0.01克），以确定身体的重量;微观尺度以确定的心脏重量精度0.001克的;称量皿;麻醉解决方案（清楚标明）;小杜瓦瓶用液氮;用冰冷的绝热容器;管和24孔板上，以收集血液和组织标本（ 表1）;外科手术器械（ 表2中，图2）;及清洁工具（ 表1）的解决方案。 </li><li> B区:安排以下项目:解剖显微镜，右心导管连接到放大器和笔记本电脑连接到压力控制单元。该导管被放置到含有水的烧杯中。安排手术器械（ 表2），最佳的长度，缝合切件，放入称重船磁带（高压釜磁带是最优的）削减到最佳的长度和宽度，为方便显微镜或实验室工作台一字排开;棉签头发卸妆液。在研究开始时，校准导管。 </li><li> C区:安排以下项目:放大镜镜片;气管插管; 1毫升注射器，缝合切到最佳长度，放入称重船小杜瓦瓶液氮;与冰的绝缘容器;管和24孔培养板，收集BAL和组织标本（ 表1）;外科手术器械（ 表2）;解决方案来执行BAL和，清洁气管插管和工具（ 表1）。 </li></ol></li></ol> 2。右心导管检查<ol><li>用一个精确的刻度称鼠标轻轻将鼠标移动到一个权衡b燕麦。请一定要轻轻地，悄悄地处理动物。记录重量。小鼠颈静脉的直径以容纳压力导管20克或更多，因为所描述的协议最适合静脉导尿较小的小鼠（17克或更多），只要是足够大的，它是可能的。 </li><li>麻醉鼠标通过注射圣阿韦坦取决于体重（10微升/克）， 例如用于20克给予200微升，25克给予250微升，30克给予300微升。的确切的注射体积取决于小鼠品系，需要进行调整。请一定要处理的动物，轻轻地，静静地，因为压力可以改变对麻醉剂的反应。 </li><li>鼠标后镇静剂，不醒人事，双折叠纸巾上。注意:在纸张上的ID号。脱毛乳液轻轻擦到腹侧方面的脖子上，以清除手术的领域。将鼠标在纸张上，等待1-2分钟。 </li><li>删除从腹侧方面​​的发头发对头发的生长方向，用棉签轻轻地抚摸着脖子上。 </li><li>请仔细确定足够的深度麻醉通过检查的脚趾反射的情况下，鼠标没有反应，捏脚趾。 </li><li>快速和轻松的工作环境的权利心脏导管插入术，其余应不超过10分钟，最佳3-6分钟。的组织，这是很重要的，因为不干燥导管需要滑行进入静脉和静脉内的水分的层上移动。一致的定时的过程中是很重要的的产生的数据组间可比性。 </li><li>将鼠标背部的纸巾，用纸巾转移到保丽龙板。修复的爪子和头，用高压釜磁带到位。 </li><li>切开皮肤，从下颚到胸骨（胸骨）。 </li><li>仔细解剖甲状腺隆重以上对下颌骨暴露右颈内静脉一第二气管。 </li><li>广场保丽龙板在解剖显微镜下按住鼠标聚焦平面在地方和镜头的约0.8倍的放大倍率（总放大倍率镜头所述目镜]约8倍。 </li><li>小心地除去周围结缔组织与手术镊子显微切割的右颈静脉。 </li><li>将两个件的缝合线的顶部的右颈静脉。领带最接近的下颌骨的缝合线，以关闭在静脉的血流量在一个狭小的涓流，放置在一个松散的结静脉周围的是最接近胸骨/乳房骨缝合线。一个可以使用的小滴的血在静脉孔后来发现，这应该成为必要。 </li><li>呼吸，放松放松颈部和下巴的肌肉，保持你的眼睛的静脉通过目镜上。这将确保你的手和手指移动安全，顺利和轻松。 ，虽然静脉用钳子在身边最亲近的下颌骨，到两者之间的缝合线用微型剪刀，用另一只手操作的静脉切开一个口子。放下手中的剪刀，并保持该手轻松，觉得自己的导管。轻轻检索拇指和食指（ 图3）之间的导管保持。将导管插入静脉的孔。 </li><li>轻轻推进右心导管插入（ 图3）。观察导管得到多远导管需要被插入的近似指数和观察显示器的标记。一旦压力曲线显示，轻轻地拧紧缝合导管周围。由于导管的抗张强度，给它一个线圈，因为鼠标的呼吸和心脏的跳动增加运动，另外用胶带固定导管。 </li><li>记录压力测量为供以后分析2分钟（ 图4）。 </li><li>打开导管缝合，然后轻轻地retrie已经导管。擦拭的部分的压力换能器的后面，将放入烧杯中，加水。请勿触摸压力传感器。 </li><li>将鼠标顶部纸巾上的区域A的安乐死，收集血液和脾组织。清洁手术器械。 </li><li>启动程序的下一个动物。当定时准许，在等待记录的压力曲线（2.15）注入麻醉。 </li></ol> 3。的补血健脾样品收集<ol><li>注射巴比妥酸盐溶液，400μL每鼠标，等待1-2分钟。这是在我们的实验室进行例行避免，小鼠可能在不经意间恢复从阿佛丁麻醉而拖垮。实验是适当的标准化取得注入相同剂量每只小鼠巴比妥盐溶液，并通过等待相同数量的时间之前，血液收获。如果允许的实验动物护理和使用委员会和只要仔细地监测作为动物麻醉深度的前放血，这个步骤可以省略。 </li><li>切口进腹。打开尾静脉（腔静脉），用移液管收集血液。 </li><li>取出脾脏，脾进入Eppendorf管内，急速冷冻在液氮中的一块，或者进入到一个含有Hanks缓冲液后准备单细胞的24孔板。 </li><li>鼠标转移之上的薄纸收集BAL肺引流淋巴结，肺，心脏组织的工作区域C。清洁手术器械。 </li></ol> 4。肺和心脏标本的收集<ol><li>解剖气管免费的肌肉和结缔组织。配售钳下的气管，拉缝合。轻轻产生足够的拉伸切开一个口子。保持温和的伸展，插入气管插管，用轻微的旋转运动，并缝合套管诠释Ø的地方。插入气管插管，气管确保湿润，如果有必要的Hanks液中加一滴。在整个过程中，呼吸，放松颈部和下巴的肌肉，保持手部动作的安全和顺畅。 </li><li>解剖开的肋骨，取出胸骨开始从腹部末端。 "请勿打扰"的胸部，因为这将使它很难找到淋巴结的内容。 </li><li>轻轻地插入Hanks缓冲液1毫升，进行支气管肺泡灌洗液（BAL），轻轻转动注射器和检索支气管肺泡灌洗液。重复3倍，接近管中，置于冰上。在整个手术过程中，保持眼睛的端部，连接到注射器的气管插管。一目了然的胸部，观察通货膨胀和通货紧缩的肺部。 </li><li>收获肺保持肋条笼形件的壁用一组镊子，寻找和检索与另一镊子的淋巴结的淋巴结。到24孔板的地点淋巴结。这是重要的为k现在要搜寻的位置:从颈部开始，找到从右侧的鼠标的肋条笼形件的入口，并期待脊柱以上（ 图5）。 </li><li>收获的心脏和纸巾。通过仔细解剖旁边的隔膜隔离的心。转移到工作区A称重，记录重量，并放入Eppendorf管在液氮中速冻。 </li><li>将关闭主支气管周围缝合，左肺叶。解剖左肺叶免费，自由的地方到eppendorf管中，急速冷冻于液氮中，或放入24孔板的井购买隔离的单细胞悬浮液中含有Hanks溶液。 </li><li>将约。 0.5毫升缓冲甲醛溶液通过气管插管到剩余的肺叶。通货膨胀后，解剖肺叶的胸部和地方50ml管中含有甲醛溶液。对于一个替代的研究设计，肺部可膨胀与优化正常切削媒体（OCT，用PBS稀释1:4）和在模具中未稀释的OCT冰冻切片后准备冻结。另一项研究设计可能需要获得快速冷冻和肺组织单细胞悬液从肺部。在这种情况下，没有通货膨胀是必要的:解剖其它肺叶;检索它们从成含有的Hanks缓冲液的24孔板的胸部和放入。 </li><li>取出气管插管，用Hanks缓冲液冲洗干净，清洁的手术器械。 </li></ol> 5。右心导管产生的压力曲线分析与没有知识中使用LabChart 7软件的组标识每个记录的记录的右心室压力数据进行分析。超过20条曲线被随机地选择和为每条曲线的最大和最小的心室压力之间的差异的测量（ΔP）。平均ΔP计算给右Ventricular收缩压。

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	<li>Price, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+pulmonary+arterial+hypertension.">Inflammation in pulmonary arterial hypertension.</a> Chest. 141, 210-221 (2012).</li><li>Olschewski, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pathophysiology+and+therapy+of+pulmonary+hypertension.">Cellular pathophysiology and therapy of pulmonary hypertension.</a> J. Lab. Clin. Med. 138, 367-377 (2001).</li><li>Hassoun, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation,+growth+factors,+and+pulmonary+vascular+remodeling.">Inflammation, growth factors, and pulmonary vascular remodeling.</a> J. Am. Coll. Cardiol. 54, S10-S19 (2009).</li><li>Rabinovitch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathogenesis+of+pulmonary+arterial+hypertension.">Molecular pathogenesis of pulmonary arterial hypertension.</a> J. Clin. Invest. 118, 2372-2379 (2008).</li><li>Steudel, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+pulmonary+hypertension+and+right+ventricular+hypertrophy+after+chronic+hypoxia+in+mice+with+congenital+deficiency+of+nitric+oxide+synthase+3.">Sustained pulmonary hypertension and right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency of nitric oxide synthase 3.</a> J. Clin. Invest. 101, 2468-2477 (1998).</li><li>Zaidi, S. H., You, X. M., Ciura, S., Husain, M., Rabinovitch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+the+serine+elastase+inhibitor+elafin+protects+transgenic+mice+from+hypoxic+pulmonary+hypertension.">Overexpression of the serine elastase inhibitor elafin protects transgenic mice from hypoxic pulmonary hypertension.</a> Circulation. 105, 516-521 (2002).</li><li>Guignabert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie2-mediated+loss+of+peroxisome+proliferator-activated+receptor-gamma+in+mice+causes+PDGF+receptor-beta-dependent+pulmonary+arterial+muscularization.">Tie2-mediated loss of peroxisome proliferator-activated receptor-gamma in mice causes PDGF receptor-beta-dependent pulmonary arterial muscularization.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 297, L1082-L1090 (2009).</li><li>West, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+hypertension+in+transgenic+mice+expressing+a+dominant-negative+BMPRII+gene+in+smooth+muscle.">Pulmonary hypertension in transgenic mice expressing a dominant-negative BMPRII gene in smooth muscle.</a> Circ. Res. 94, 1109-1114 (2004).</li><li>Cook, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+eNO+and+pulmonary+iNOS+expression+in+eNOS+null+mice.">Increased eNO and pulmonary iNOS expression in eNOS null mice.</a> Eur. Respir. J. 21, 770-773 (2003).</li><li>West, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+expressing+BMPR2R899X+transgene+in+smooth+muscle+develop+pulmonary+vascular+lesions.">Mice expressing BMPR2R899X transgene in smooth muscle develop pulmonary vascular lesions.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 295, L744-L755 (2008).</li><li>Tu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+fibroblast+growth+factor-2+signaling+contributes+to+altered+endothelial+phenotype+in+pulmonary+hypertension.">Autocrine fibroblast growth factor-2 signaling contributes to altered endothelial phenotype in pulmonary hypertension.</a> Am. J. Respir. Cell Mol. Biol. 45, 311-322 (2011).</li><li>Daley, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+arterial+remodeling+induced+by+a+Th2+immune+response.">Pulmonary arterial remodeling induced by a Th2 immune response.</a> J. Exp. Med. 205, 361-372 (2008).</li><li>Song, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation,+endothelial+injury,+and+persistent+pulmonary+hypertension+in+heterozygous+BMPR2-mutant+mice.">Inflammation, endothelial injury, and persistent pulmonary hypertension in heterozygous BMPR2-mutant mice.</a> Am. J. Physiol. Heart Circ. Physiol. 295, 677-690 (2008).</li><li>Thibault, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+assessment+of+murine+pulmonary+arterial+pressure:+validation+and+application+to+models+of+pulmonary+hypertension.">Noninvasive assessment of murine pulmonary arterial pressure: validation and application to models of pulmonary hypertension.</a> Circulation. Cardiovascular imaging. 3, 157-163 (2010).</li><li>Otto, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+hypertension+and+right+heart+failure+in+pituitary+adenylate+cyclase-activating+polypeptide+type+I+receptor-deficient+mice.">Pulmonary hypertension and right heart failure in pituitary adenylate cyclase-activating polypeptide type I receptor-deficient mice.</a> Circulation. 110, 3245-3251 (2004).</li><li>Burton, V. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+leukocyte+recruitment+via+CXCR1/2+inhibition+stops+the+progression+of+PAH+in+mice+with+genetic+ablation+of+endothelial+BMPR-II.">Attenuation of leukocyte recruitment via CXCR1/2 inhibition stops the progression of PAH in mice with genetic ablation of endothelial BMPR-II.</a> Blood. 118, 4750-4758 (2011).</li><li>Fujita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+hypertension+in+TNF-alpha-overexpressing+mice+is+associated+with+decreased+VEGF+gene+expression.">Pulmonary hypertension in TNF-alpha-overexpressing mice is associated with decreased VEGF gene expression.</a> J. Appl. Physiol. 93, 2162-2170 (2002).</li><li>Motley, H. L., Cournand, A., Werko, L., Himmelstein, A., Dresdale, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influence+of+Short+Periods+of+Induced+Acute+Anoxia+Upon+Pulmonary+Artery+Pressures+in+Man.">The Influence of Short Periods of Induced Acute Anoxia Upon Pulmonary Artery Pressures in Man.</a> Am. J. Physiol. 150, 315-320 (1947).</li><li>Liljestrand, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Pulmonary+Arterial+Blood+Pressure.">Regulation of Pulmonary Arterial Blood Pressure.</a> Arch. Intern. Med. 81, 162-172 (1948).</li><li>Euler, U. S. V., Liljestrand, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+pulmonary+arterial+blood+pressure+in+the+cat.">Observations on the pulmonary arterial blood pressure in the cat.</a> Acta Physiol. Scand. 12, 301-320 (1946).</li><li>Van den Broeck, W., Derore, A., Simoens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+nomenclature+of+murine+lymph+nodes:+Descriptive+study+and+nomenclatory+standardization+in+BALB/cAnNCrl+mice.">Anatomy and nomenclature of murine lymph nodes: Descriptive study and nomenclatory standardization in BALB/cAnNCrl mice.</a> Journal of immunological. 312, 12-19 (2006).</li><li>Rabinovitch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II+prevents+hypoxic+pulmonary+hypertension+and+vascular+changes+in+rat.">Angiotensin II prevents hypoxic pulmonary hypertension and vascular changes in rat.</a> Am. J. Physiol. 254, 500-508 (1988).</li><li>Rabinovitch, M., Gamble, W., Nadas, A. S., Miettinen, O. S., Reid, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+pulmonary+circulation+after+chronic+hypoxia:+hemodynamic+and+structural+features.">Rat pulmonary circulation after chronic hypoxia: hemodynamic and structural features.</a> Am. J. Physiol. 236, 818-827 (1979).</li><li>Rabinovitch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+pulmonary+blood+flow+affect+vascular+response+to+chronic+hypoxia+in+rats.">Changes in pulmonary blood flow affect vascular response to chronic hypoxia in rats.</a> Circ. Res. 52, 432-441 (1983).</li><li>Kugathasan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+angiopietin-1-Tie2+pathway+prevents+rather+than+promotes+pulmonary+arterial+hypertension+in+transgenic+mice.">The angiopietin-1-Tie2 pathway prevents rather than promotes pulmonary arterial hypertension in transgenic mice.</a> J. Exp. Med. 206, 2221-2234 (2009).</li><li>Bearer, C., Emerson, R. K., ORiordan, M. A., Roitman, E., Shackleton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+tobacco+smoke+exposure+and+persistent+pulmonary+hypertension+of+the+newborn.">Maternal tobacco smoke exposure and persistent pulmonary hypertension of the newborn.</a> Environ. Health Persp. , 105-202 (1997).</li><li>Graham, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis-induced+experimental+pulmonary+hypertension:+role+of+interleukin-13+signaling.">Schistosomiasis-induced experimental pulmonary hypertension: role of interleukin-13 signaling.</a> Am. J. Pathol. 177, 1549-1561 (2010).</li><li>Butrous, G., Ghofrani, H. A., Grimminger, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+vascular+disease+in+the+developing+world.">Pulmonary vascular disease in the developing world.</a> Circulation. 118, 1758-1766 (2008).</li><li>Crosby, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Praziquantel+reverses+pulmonary+hypertension+and+vascular+remodeling+in+murine+schistosomiasis.">Praziquantel reverses pulmonary hypertension and vascular remodeling in murine schistosomiasis.</a> Am. J. Respir. Crit. Care Med. 184, 467-473 (2011).</li></ol>

没有利益冲突的声明。

此处列出的实验流程，允许快速和同时测量右心室收缩压和收获的样品分析的反应在肺部，心脏和免疫系统的小鼠。该过程结合心脏生理学测量，微解剖和随后的组织为活细胞研究，组织学分析，或组学分析的组织收获。的完整过程只需不到20分钟，每鼠标。由于工作区域组织的工作流程，2-3的动物可以同时进行研究。因此，程序是适用于小的，有针对性的实验12和设置在很宽的范围内进行的大型屏幕，从一个小型实验室，大型制药研究。 心脏生理测量和组织收获相结合，将打开的可能性进行分析，检测生物网络控制或同步心脏，肺一ND免疫功能，利用转基因和KO小鼠资源。同时程序流程的一个重要优点是在每个研究所需的动物的数目减少。这个程序的工作流程中的另一个应用是研究其他的或额外的相同的动物的器官的可能性。 这里列出的程序产生对心脏的压力非常迅速，在5-10分钟的麻醉诱导。这使得研究的动物，而他们自发地呼吸室内空气。一个限制的做法是，它是侵入性的:动物麻醉，他们被安置到自己的背上，传递的心房进入右心室，通过颈静脉导管。该限制是此过程的优点，因为该数据表示随着时间的推移在右心室的压力变化的直接测量。进一步的技术改进，特别是依靠小型化的公关essure导管，可允许，放置永久，留置导管在小鼠体内，使肺动脉压力的直接记录在几个星期的时间，22日至24日在大鼠和其他大型动物正在执行。如果插入的心导管通过颈内静脉紧密的胸部，自主呼吸的动物是不可能的，另一种记录的右心室收缩压的变化，可在通风良好的动物，通过开放的胸部，并把压力通过导管切口直接进入右心室25。可以采用超声心动图右心功能的非侵入性测量。此过程可以进行右心导管检查前，或多次研究疾病恶化之前的侵入性测量5,14-17小鼠可以通过超声心动图检查。 此过程中的另一种应用是，以获得额外的l数据。作为一个例子，使用可以测量流量和压力的导管，右心脏输出可以同时一起记录与右心的压力变化。这里所概述的程序也可以被用来进一步理解的生理，细胞和分子的变化触发免疫反应以外的肺动脉高压，例如，的遗传突变8,10，香烟烟雾暴露26，或微生物感染27-29 。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td> 2 - 甲基-2 - 丁醇</td><td> Sigma-Aldrich公司</td><td> 152463 </td><td></td></tr><tr><td> 2,2,2 - 三溴乙醇</td><td> Sigma-Aldrich公司</td><td> T48402 </td><td></td></tr><tr><td>消毒肥皂（专题喷雾TB加STERIS） </td><td> Fisher Scientific则</td><td> 1629至1608年</td><td></td></tr><tr><td> 200的证明，乙醇，绝对无水ACS / USP级</td><td> PHARMCO-AAPER </td><td> 111000200 </td><td>用蒸馏水稀释至70％的</td></tr><tr><td>甲醛溶液</td><td> Sigma-Aldrich公司</td><td> F1635-500ML </td><td>稀释至7-10％，预计ldehyde浓度在PBS中浓度为1x使用PBS原液和水</td></tr><tr><td> Hanks溶液，无钙，镁</td><td> Fisher Scientific则</td><td> 21-022-CV </td><td></td></tr><tr><td> OCT </td><td>组织康</td><td> 4583 </td><td></td></tr><tr><td> （10,000 U / ml）的青霉素/链霉素（10000毫克/毫升）解决方案</td><td> Thermo Scientific的</td><td> SV30010 </td><td></td></tr><tr><td>磷酸盐缓冲盐水（PBS），无钙，无镁，1倍和10倍的解决方案</td><td> Fisher Scientific则</td><td></td><td></td></tr><tr><td>戊巴比妥钠26％ </td><td> Fort Dodge动物保健</td><td> NDC 0856-0471-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> 医学实验 </td></tr><tr><td>板12，24，96以及</td><td>鹰</td><td></td><td></td></TR&gt; <tr><td>移液管</td><td> Fisher Scientific则</td><td> 13-711-9BM </td><td></td></tr><tr><td>管，EDTA涂层; </td><td>萨尔斯塔特</td><td> 2013-08 </td><td></td></tr><tr><td>管0.65毫升和1.7毫升的微离心</td><td> VWR </td><td></td><td></td></tr><tr><td>管12×75毫米聚丙烯</td><td> Fisher Scientific则</td><td> 14-956-1D </td><td></td></tr><tr><td>管，各种大小，聚丙烯</td><td> Fisher Scientific则</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 仪器 </td></tr><tr><td>钳，DUMON第5精细</td><td>精细科学工具</td><td> 11254-20 </td><td></td></tr><tr><td>钳，超等格雷夫-0.5mm的技巧弯曲</td><td>精细科学工具</td><td> 11152-10 </td><td></td></tr><tr><td>钳，额外的网络连接东北格雷夫-0.5mm的提示直</td><td>精细科学工具</td><td> 11150-10 </td><td></td></tr><tr><td> 19 GA 18GA，套管</td><td> BD </td><td>精密的滑翔针</td><td>切到最佳长度，减弱外嘶哑创建一个粗糙的外表面</td></tr><tr><td>剪刀，剪刀剥离超薄刀片9厘米</td><td>精细科学工具</td><td> 14081-09 </td><td></td></tr><tr><td> BAL，编织丝线缝合，缝合4-0 </td><td>精细科学工具</td><td> SP116 </td><td></td></tr><tr><td>缝合右心导管检查，编织丝线缝合，6-0 </td><td>泰利福医疗</td><td> 18020-60 </td><td></td></tr><tr><td>注射器，1毫升</td><td> BD </td><td> 309659 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>放大器，PowerLab系统4/30 </TD&gt; <td> ADInstrument </td><td>型号ML866 </td><td></td></tr><tr><td>导管，压力F1.4 </td><td>米勒仪器公司</td><td> 840-6719 </td><td></td></tr><tr><td>解剖显微镜</td><td>彩色摄影</td><td></td><td></td></tr><tr><td>钳，Vannas春风似剪刀毫米刀片</td><td>精细科学工具</td><td> 15000-00 </td><td></td></tr><tr><td>卤素照明台放大镜</td><td> Fisher Scientific则</td><td> 11-990-56 </td><td></td></tr><tr><td>笔记本电脑</td><td>华硕</td><td>型号A52F酷睿i5处理器，15英寸</td><td></td></tr><tr><td>光源</td><td> Amscope </td><td> HL-250-A </td><td></td></tr><tr><td>压力控制单元</td><td>米勒仪器公司</td><td> PCU-2000 </td><td></td></tr><tr><td>软件，Labchart。亲V.7 </td><td> AD在剂量率仪</td><td></td><td></td></tr></tbody></table>

right ventricular systolic pressure measurements in combination with harvest of lung and immune tissue samples in mice

正确的心脏的功能是把血液通过肺部，从而将右心脏的生理和肺血管生理。炎症的心脏和肺的功能，是一种常见的改性剂，通过制定生产的细胞因子和生长因子，细胞浸润，并开始重塑过程1。 右心室与左心室相比，是一个低压泵工作在一个相对窄的区域的压力变化。增加与肺动脉压力增加的压力在肺血管床和肺动脉高压2。肺动脉高压往往伴有炎症性肺部疾病，如慢性阻塞性肺疾病，自身免疫性疾病3。由于肺动脉高压赋予预后不良的生活质量和寿命，大量的研究是针对了解的机制，mig的HT药物干预4的目标。有效的管理手段，为肺动脉高压的发展面临的主要挑战仍然是复杂的分子和细胞变化在权利的心，肺和免疫系统的同时了解。 在这里，我们提出了一个程序的工作流程，快速，精确的测量压力变化在正确的小鼠心脏的心脏，肺和免疫组织样本的同时收获。该方法是基于直接导管右心室，通过颈内静脉接近上身的小鼠，最早是在20世纪90年代后期，作为替代措施的压力在肺动脉5-13。组织团队的方式有利于快速右心导管检查技术。这使得能够进行测量的小鼠的自发呼吸室内空气。在不同的工作领域的组织的工作流程减少了时间延迟和打开的可能性，同时进行生理实验和收获的免疫系统，心脏和肺组织。 这里列出的程序的工作流程，可以适应各种各样的实验室设置和研究设计，从小型的，有针对性的实验，到大型药物筛选试验。同时采集心肌的生理数据，可以扩展到包括超声心动图5,14-17和收获的心脏，肺和免疫组织减少了需要获得数据的科学知识为基础向前移动的动物。这里介绍的程序的工作流程也提供了理想的基础免疫，肺和心脏功能的网络链接获取知识。这里概述的相同的原则可以适用于研究根据需要的其它的或附加的器官。

获得合适的心脏压力曲线的主要结果是通过合适的心脏导管的正确位置。右心室的导管内部的正确放置的压力时间曲线的形状是重要的，因为会导致压力高原（ 图4）。相反，高低不平的曲线，表明由呼吸或心脏跳动的运动靠在墙上的右心室导管移动。为了检测的阶段的动物的生存的潜在的问题，需要计算ΔP，和心脏的速度的标准偏差。预计这两个值都没有显着不同的治疗组之间。此外，期望的是，ΔP值和心脏的速度并没有显示整个记录时间的漂移。例如，ΔP值缓慢增加，表明缺氧性肺血管收缩18-20。降低心脏率indicatE的麻醉水平过高导致死亡的动物。在我们的实验室中，不同的人执行的程序。在所有组小鼠中，我们经常达到90％-95％的成功率获得右心室压力数据。 在学习的过程，它是最好的开始年龄较大的雌性小鼠（25-30克），有较大的颈静脉，往往有减少皮下脂肪沉积比雄性小鼠。的最佳方式，以确定导管的放置是安乐死动物尽快的内部被固定的静脉导管。解剖动物，从心开始直接可视化的导管的位置。这将极大地促进问题的解决。 获得支气管肺泡灌洗液的第一个结果是气管插管的正确位置。这是由Hanks缓冲液注入肺部的通胀，通缩的肺部当BUFF呃被除去。复苏的预期是在控制动物的注入量的70％至80％，小于（50％），肺部有炎症的动物。泡沫顶部回收的BAL液表示表面活性剂的存在下。回收的BAL液中的血液污染发生，因为显着的肺部炎症的存在下，或者，因为灌注和恢复的清洗流体是太快。值得注意的是，一些急性内的肺损伤的措施，可诱发的BAL过程。如果设计的实验，来了解这些急性损伤参数和BAL检查也是很重要的，然后需要一个肺叶被束缚和删除前进行BAL。 在此处显示的过程中，我们收获的肺组织进行组织学的肺部没有标准化的通货膨胀和灌注。确切的病理形态计量学的主要读出的情况下，应该被夸大，肺部通过气管，PErfusion应通过肺动脉，所有标准化的压力下进行。 在协议中，心脏的解剖提供富尔顿指数，作为衡量右心肥大的右心室/左心室和室间隔重量（重量）。此外，新的方法正在开发为基础的组织学评价的权利的心。新兴右心肥大的组织措施一）的核数定义的区域内随机选择合适的心脏，和b）计数的血管，肝纤维化指标的单位面积的心。 在我们的实验室中，正确的恢复肺引流纵隔和气管支气管淋巴结（ 图5）经常通过流式细胞仪检测验证，因为这些组织是非常小的对照组。然而，依赖于微生物组在壳体设施和动物的年龄，大小的淋巴结中unchallenGED，对照组小鼠可能会​​足够大，只有视觉确认。当验证通过流式细胞术，淋巴结细胞的胸腺细胞区别开来。因事故，可以很容易地被采样胸腺与淋巴结一起因为胸腺和淋巴结位于相互接近的，并且包含在肋条笼形件。这种区别是，胸腺靠近乳房骨和靠近脊柱的淋巴结。进一步的挑战是，胸腺是非常大的，含有更多的细胞比小淋巴结。胸腺细胞可以很容易地区别于淋巴结细胞，使用流式细胞仪和CD3，CD4，CD8标记。淋巴结有典型的CD3 +细胞，其中大部分是CD4或CD8阳性。相反，大部分的胸腺细胞是阳性的所有三个标记（CD3 +，CD4 +，CD8 +）。此外，鼠标淋巴结的本地化深入的信息可以发现的手稿面包车登布洛克等 21。 各主要程序步骤（心脏导管插入术，恢复血液和脾，收获的的BAL，肺和心脏组织）是一致的计时产生的数据，使治疗组之间比较强劲的关键。因此，它是至少两个，和三个最好的建议中，研究者协作完成实验。此外，重要的是，调查人员在执行过程没有知识的群体特征的动物。因此，确定的数据具有掩盖的组标识的方式进行。最后，它也是很重要的分析，其中动物的顺序是随机的。例如，一个简单的标识符是动物实验和连续编号的日期。例如如果有4组动物（AD），指定的鉴定与研究的动物按以下顺序:DATE_1:AA，_2:dat​​e_3:钡，钙，date_4:大date_5:从头date_6:BB，date_7:CB，等等。 本实验的流量控制肺部炎症，右心功能的分子机制研究的深入，同时允许。的同时进行数据和样品收集到分子网络中实现更好的见解，并需要以获得新的生物学知识的动物的数目减少。 <table border="1"><tbody><tr><td> 管/杯的类型 </td><td> 溶液（体积） </td><td> 采用 </td></tr><tr><td colspan="3"> 血的处理 </td></tr><tr><td> Eppendorf公司，1.5毫升; </td><td></td><td>血的血清</td></tr><tr><td> EDTA涂层2.0毫升</td><td></td><td>血的血浆</td></tr><tr><td> Eppendorf公司，0.5毫升; </td><td></td><td>冷冻的血清或血浆</td></tR&gt; <tr><td>处理BAL </td><td></td><td></td></tr><tr><td>聚丙烯，圆底，4.5毫升; </td><td></td><td>收集支气管肺泡灌洗液</td></tr><tr><td>聚丙烯，圆底，4.5毫升; </td><td></td><td>冻结BAL上清液</td></tr><tr><td> 96孔板中，掠过</td><td></td><td> BAL细胞</td></tr><tr><td colspan="3"> 组织处理 </td></tr><tr><td>聚丙烯，50毫升; </td><td>甲醛，7.5毫升</td><td>修复肺组织</td></tr><tr><td> Eppendorf公司，1.5毫升; </td><td></td><td>急速冷冻肺叶</td></tr><tr><td> Eppendorf公司，1.5毫升; </td><td></td><td>速冻合适的心脏</td></tr><tr><td> Eppendorf公司，1.5毫升; </td><td></td><td>速冻左心脏</td></tr><tr><td> 24 - 孔板</td><td> Hanks缓冲液，0.5〜1毫升</td><td>快速存储的肺叶后，我的染料溶液的单细胞</td></tr><tr><td> 24 - 孔板</td><td> Hanks缓冲液，0.5〜1毫升</td><td>快速存储脾脏后隔离的单细胞</td></tr><tr><td> 24 - 孔板</td><td> Hanks缓冲液，0.5〜1毫升</td><td>快速存储隔离的单细胞后淋巴结</td></tr><tr><td colspan="3"> 对于程序 </td></tr><tr><td>玻璃烧杯中，将300毫升</td><td>无菌水</td><td>保湿和清洁导管</td></tr><tr><td>聚丙烯管，50毫升; </td><td> Hanks缓冲液，50毫升</td><td>执行BAL </td></tr><tr><td>聚丙烯管，50毫升; </td><td> Hanks缓冲液，50毫升</td><td>洗气管插管</td></tr><tr><td>聚丙烯管，50毫升; </td><td>消毒液， </td><td>清洁工具</td></tr><tr><td>聚丙烯管，50毫升; </td><td> 70％乙醇</td><td>氯eaning仪器</td></tr><tr><td>聚丙烯管，50毫升; </td><td>自来水</td><td>清洁工具</td></tr></tbody></table> 表1中。下游和组织的管和解决方案。 <table border="1"><tbody><tr><td> 仪器 </td><td> 描述 </td><td> 使用 </td></tr><tr><td>剪刀</td><td>圆形或直5.5英寸，圆形或尖锐的两端</td><td rowspan="4"> A区</td></tr><tr><td>剪刀</td><td>圆形的4.0英寸，末端锐利</td></tr><tr><td>镊子</td><td> 4.5英寸，弯曲，扁平，抓两端</td></tr><tr><td>镊子</td><td> 4.0英寸，弯曲，扁平，抓两端</td></tr><tr><td>剪刀</td><td>的超薄Blades/Angled-9厘米</td><td rowspan="7"> B区</td></tr><tr><td>微型剪刀</td><td>货车NAS春风似剪刀 - 2毫米刀片</td></tr><tr><td>镊子</td><td>钳（DUMON第5精细） </td></tr><tr><td>镊子</td><td> 4.0英寸，弯曲，扁平，抓两端</td></tr><tr><td>镊子</td><td> 4.0英寸，直，平，抓两端</td></tr><tr><td>缝合</td><td>编织丝线缝合6-0 </td></tr><tr><td>导管</td><td>压力导管</td></tr><tr><td>剪刀</td><td>圆形或直5.5英寸，圆形或尖锐的两端</td><td rowspan="6"> C区</td></tr><tr><td>剪刀</td><td>圆形的4.0英寸，末端锐利</td></tr><tr><td>镊子</td><td> 4.5英寸，弯曲，扁平，抓两端</td></tr><tr><td>镊子</td><td> 4.0英寸，弯曲，扁平，抓两端</td></tr><tr><td>套管</td><td>气管插管</td></tr><tr><td>缝合</td><td>编织丝线缝合4-0 </td></tr></tbody></table> 表2。组织的工具，缝线，导尿管，气管插管。 <img src="/files/ftp_upload/50023/50023fig1.jpg" alt="图1"/> 图1。略图A）工作区的工作区。一个记录，体重，血液和脾组织收集和临时存放空间的动物。 B）工作区乙右心导管C）工作BAL，肺，肺癌的淋巴结和心脏组织，收获面积为C。 <img src="/files/ftp_upload/50023/50023fig2.jpg" alt="图2"/> 图2的过程中使用的仪器。A）仪器工作区域A; B）右心导管检查（工作区B）的工具; C）插件文书的工作区C，D）气管插管。的标尺显示的大小的仪器。 <img src="/files/ftp_upload/50023/50023fig3.jpg" alt="图3"/> 图3右心脏导管。右心导管显示的商标和磁带（A），或持有之手（B）。铅笔的点，在导管上的标记（A）。导管插入（CE）:清洗的右颈静脉（C），，缝合静脉用钳子举行，导管被推进先前制成的静脉（D）朝向一个孔;导管插入后的静脉（E）。箭头指向的导管（D，E）。 <img src="/files/ftp_upload/50023/50023fig4.jpg" alt="图4" fo:content-width="4in" fo:src=<html> "机构/ files/ftp_upload/50023/50023fig1highres.jpg"/&gt; 图4。在右心的压力变化的示例跟踪。的痕迹，显示压力变化（毫米汞柱）随时间（分:秒），该软件显示的曲线在两个不同的速度。的痕迹（A）控制鼠标和鼠标与炎症引起的肺小动脉重构和高血压（B）所示。用于右心室收缩压力的测量的曲线被高亮显示。请注意，清晰的压力高原中高亮显示的曲线。箭头指向的曲线，有一个尖峰，表示一个技术性错误。这些高低不平的曲线不能用于测量右心室收缩压， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/50023/50023fig4large.jpg" target="_blank">点击此处查看大图</a> 。 0023fig5.jpg"ALT ="图5"/&gt; 图5。相对于肋骨和脊柱的纵隔/气管支气管淋巴结的位置。箭头指向的淋巴结。

Watch this Scientific Journal Video about Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice at JoVE.com

右心室收缩压测量与小鼠肺和免疫组织样本的采集相结合|免疫学和感染|视频

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

一个特异，快速的协议，同时探讨合适的心脏功能，肺部炎症和免疫反应，被描述为一种学习工具。视频和数字，描述的生理和显微切割技术在组织团队的方法，是适用于用于小型到大型研究。

右心室收缩压测量组合在小鼠的肺和免疫组织样品的收获

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

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Circulatory and Pulmonary Systems

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25.1K 次观看.  New York University School of Medicine, Tuxedo. 一个特异，快速的协议，同时探讨合适的心脏功能，肺部炎症和免疫反应，被描述为一种学习工具。视频和数字，描述的生理和显微切割技术在组织团队的方法，是适用于用于小型到大型研究。

视频: Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable around embryonic day (E) 8.51. Systolic left ventricular (LV) pressure is 2 mmHg at E9.5 and 11 mmHg at E14.52. At these mid-embryonic stages, the LV is clearly visible through the chest wall for invasive pressure measurements because the ribs and skin are not fully developed. Between E14.5 and birth (approximately E21) imaging methods must be used to view the LV. After birth, mean arterial pressure increases from 30 - 70 mmHg from postnatal day (P) 2 - 353. Beyond P20, arterial pressure can be measured with solid-state catheters (i.e. Millar or Scisense). Before P20, these catheters are too big for developing mouse arteries and arterial pressure must be measured with custom pulled plastic catheters attached to fluid-filled pressure transducers3 or glass micropipettes attached to servo null pressure transducers4.
Our recent work has shown that the greatest increase in blood pressure occurs during the late embryonic to early postnatal period in mice5-7. This large increase in blood pressure may influence smooth muscle cell (SMC) phenotype in developing arteries and trigger important mechanotransduction events. In human disease, where the mechanical properties of developing arteries are compromised by defects in extracellular matrix proteins (i.e. Marfan's Syndrome8 and Supravalvular Aortic Stenosis9) the rapid changes in blood pressure during this period may contribute to disease phenotype and severity through alterations in mechanotransduction signals. Therefore, it is important to be able to measure blood pressure changes during late embryonic and neonatal periods in mouse models of human disease.
We describe a method for measuring LV pressure in late embryonic (E18) and early postnatal (P1 - 20) mice. A needle attached to a fluid-filled pressure transducer is inserted into the LV under ultrasound guidance. Care is taken to maintain normal cardiac function during the experimental protocol, especially for the embryonic mice. Representative data are presented and limitations of the protocol are discussed.

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

测量中晚期胚胎和新生小鼠左心室压力

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable ...

科研

生物工程

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

右心小鼠的超声心动图评价

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

医学

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

右心室结构和功能的肺动脉缩窄经胸超声小鼠模型的评估

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

在小鼠肺压力容积曲线的测量

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease. 
Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.

Pulmonary arterial hypertension (PAH) is a disease of pulmonary arterioles that leads to their obliteration and the development of right ventricular failure. Rodent models of PAH are critical in understanding the pathophysiology of PAH. Here we demonstrate hemodynamic characterization, with right heart catheterization and echocardiography, in the mouse and rat.

肺动脉高压的啮齿动物模型的血流动力学特性

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

表征隔离，通风，和仪表小鼠肺灌注与脉动流

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

生物学

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

小鼠右心室舒张参数的超声心电图测量

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart&#160;catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to&#160;treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right&#160;ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented.

使用缺氧/SU5416模型对小鼠的肺高血压进行诱导和特征

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

小鼠右心室系统及缺氧诱发肺动脉高血压的侵入性血液动力学评估

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, there is no cure for PAH. It is important to discover new compounds that can be used to treat PAH. The mouse hypoxia-induced PAH model is a widely used model for PAH research. This model recapitulates human clinical manifestations of PAH Group 3 disease and is an important research tool to evaluate the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds in mice. For a compound that needs to be given directly into the bloodstream, optimizing intravenous (IV) administration is a key part of the experimental procedures. Ideally, the IV injection system should permit multiple injections over a set time course. Although the mouse hypoxia-induced PAH model is very popular in many laboratories, it is technically challenging to perform multiple IV bolus dosing and invasive hemodynamic assessment in this model. In this protocol, we present step-by-step instructions on how to carry out multiple IV bolus dosing via mouse jugular vein and perform arterial and right ventricle catheterization for hemodynamic assessment in mouse hypoxia-induced PAH model.

This protocol provides a step-by-step procedure for executing multiple intravenous bolus dose administration and invasive hemodynamic monitoring in mice. Investigators can use this protocol for future therapeutic compound screening for pulmonary artery hypertension.

缺氧诱导小鼠肺动脉高压模型中的多次静脉推注给药和侵入性血流动力学评估

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, ...