This work was supported by NIH grants HL-089779 (DW), HL-112270 (KAP) and HL-102836 (KAP) and Cimphoni Life Sciences (part of DW salary). The authors thank Meghann Sytsma for editing the manuscript.

本研究是在严格按照指南中的建议，美国国立卫生研究院的实验动物的护理和使用进行。该协议被批准的机构动物护理和使用委员会（协议:AUA＃1517）。所有涉及小鼠的研究是与小灵通的政策相一致进行。 1.诱饵肽的设计<ol><li>找到IRF5的3D结构，立足于它的设计。设计一个17聚体，称为IRF5D（ELDWDADDIRLQIDNPD），其中，天门冬氨酸（D）取代为丝氨酸（S）在421模仿磷酸化IRF5 - 438（ELSWSADSIRLQISNPD）。 3D巯基和激活IRF5的机制的认识， 即 ，丝氨酸磷酸化，分析表明，一种策略靶向IRF5是创造一个诱饵肽。 见图 1。以确定在三维结构的最佳区域，可使用分子建模软件设计小moleculES和肽15。 注意:在此过程中的关键步骤是目标蛋白质的三维结构和其可能的结合位点的可用性。 IRF5的三维结构，准确地反映后，磷酸化如何IRF5二聚化。门冬氨酸丝氨酸或苏氨酸的替代是外包。该肽不被链接到IRF5单独合成。 </li><li>简单地通过用基于原序列S.D。复制磷酸域（ELSWSADSIRLQISNPD，使与AC-ELDWDADDIRLQIDNPD-NH 2（IRF5D）的氨基酸序列的新的肽IRF5S。 </li></ol> 2.生物层干涉（BLI） 注:重组IRF5纯化是外包的。 16 <ol><li>生物素标记IRF5以1:1的摩尔比:1的生物素与掺入长链连接基生物素化试剂配体。长链连接器将确保一个更加积极和同质西甲ND面。 <ol><li>加入1毫升2毫克/毫升IRF5至20mM生物素试剂的13微升或如果需要增加体积。在冰上孵育2小时。标记后，可使用脱盐离心柱以从标记蛋白去除反应混合物。 </li></ol></li><li>孵育与生物传感器15分钟的生物素标记的配体。避免过度标记和调整为1:1的摩尔比。标记后，可使用脱盐离心柱以从标记蛋白去除反应混合物。 </li><li>孵育生物素标记IRF5生物传感器在不同浓度的IRF5诱饵肽10分钟。结合和解离速率来确定如所述10，17，18。 </li></ol> 3.细胞凋亡与增殖检测<ol><li> 通过四氮唑的生物还原增殖试验 <ol><li>辖IRF5D稀释在PBS中（1毫克/公斤/天，在20 G鼠标通过一个标准的洁肤技术或通过单独的PBS注入量20μL）皮下注射27号针头啧/ +和C57BL / 6J小鼠21天。 </li><li>麻醉用异氟烷（5％）的小鼠，并执行一个颈脱位。通过切除切开胸部沿着胸骨的心。提起用钳子心脏，取出心脏。 </li><li>裂解用含有50mM的Tris-HCl pH 7.4的，通过Bradford测定法19 0.5％的NP-40，250 mM氯化钠，5mM的EDTA，50mM的氟化钠10和确定蛋白质浓度的蛋白质裂解缓冲液的心。 </li><li>以如在第7节中所述分离之C57B1 / 6J心脏矩阵涂层96孔板稀释的PBS悬浮之C57B1 / 6J心脏基质至20微克/ mL的浓度，并添加之C57B1 / 6J心脏到每个30微升良好。让它在孵化器在37℃沉降过夜。 </li><li>使用500毫升的内皮细胞基础米培养人脐静脉细胞EDIA用0.4％牛脑提取物，0.1％抗坏血酸，0.1％氢化可的松，0.1％的人表皮生长因子，2％胎牛血清和0.1％庆大霉素/两性霉素B添加到它。培养上在5％的CO 2和95％的室内空气，每孔25,000个细胞的密度96孔板的细胞。与0之间增加IRF5D的浓度刺激 - 100微克/毫升到介质（体积为0和50微升/毫升之间）。 </li><li>确定细胞增殖三天后和评估酶标仪吸光度差异。该反应的MTS四唑鎓化合物的生物还原。 NADPH或NADH被在代谢活性细胞的脱氢酶产生的。 </li><li>作为在MTS四唑鎓化合物被储存在-20℃下冷冻，解冻化合物在室温下慢慢地在约90分钟。吸取20微升到96孔板的各孔中，培养基中添加100微升到它。在37℃下孵育板用于在腐殖1至4小时dified，5％的CO 2室中。 </li><li>在490纳米的波长读取吸光度。 </li></ol></li><li> 通过 caspase活性细胞凋亡检测 注:实验装置是与上述相同的3.1.1至3.1.5。 <ol><li>刺激细胞用0之间增加IRF5D浓度 - 100微克/毫升到介质。 </li><li>添加绿检测到胱天蛋白酶3/7衬底的内皮细胞，并在37°C孵育它为30分钟。同的五百三十零分之五百〇二纳米的吸收/发射波长同时评估对酶标仪的荧光。一式三份运行实验。 注:绿色荧光剂是结合到核酸结合染料的4个氨基酸的肽。当胱天蛋白酶3和7的激活后裂解该试剂变成荧光。该荧光被测量。它的优点是在洗涤步骤的减少。 </li></ol></li></ol> 4.在听到IRF5和ICAM-1表达的评估ŧ <ol><li>通过一个标准的洁肤技术或PBS单独啧/ +和C57BL / 6J小鼠一次是用27号针头管理IRF5D在PBS稀释（1毫克/公斤/天，在20 G鼠标注入量20μL）皮下注射每天，持续21天。 </li><li>麻醉用异氟烷（5％）的小鼠，并执行一个颈脱位。通过切除切开胸部沿着胸骨的心。提起用钳子心脏，取出心脏。 </li><li>裂解用含有50mM的Tris-HCl pH 7.4的，通过Bradford测定法19 0.5％的NP-40，250 mM氯化钠，5mM的EDTA，50mM的氟化钠10和确定蛋白质浓度的蛋白质裂解缓冲液的心。 <ol><li>在心中的裂解液进行免疫印迹。稀释样品至25微克总蛋白和40微升用含有5％巯基乙醇的Laemmli样品缓冲液的体积。在50毫伏15％TGX凝胶5分钟 - 运行在一个4样本。加大电压为100 mV的1小时，直到染料前沿耗尽。</li><li>放置在1×转移缓冲液的凝胶（25毫摩尔Tris，190毫甘氨酸，20％甲醇，如果需要的话调节pH至8.3），并孵育10 - 15分钟。 </li><li>装配转移三明治（海绵，过滤器，胶，纤维素膜，过滤器，海绵）。确保无气泡被困在中间。印迹应当是在阴极和阳极上的凝胶。在寒冷的房间在100的mA 1小时。 </li><li>第二天，漂洗印迹，然后方框在5％脱脂奶粉的背景，在室温下30分钟。冲洗用Tris印迹缓冲含有5％吐温三次盐水，每次5分钟。 </li><li>用初级多克隆抗体IRF5或ICAM-1的第一抗体孵育过夜。稀释在Tris抗体以1缓冲盐水:1000稀释。 </li><li>稀释在Tris二次抗体缓冲盐水孵育印迹在室温下1小时。对于二级抗体，使用辣根过氧化物酶偶联的驴抗小鼠IgG（1:10,000）和辣根过氧化物酶的驴抗山羊IgG（1:10,000）分别。 </li><li>应用化学发光的印迹根据制造商的协议混合组分后，孵育3分钟以发光的感兴趣的频带。揭露印迹X射线胶片。使用ImageJ的测量密度。正常化的密度值向控制带10。 </li></ol></li></ol> 5. IRF5后抑制炎症细胞数的评估<ol><li>通过标准洁肤技术或通过PBS施用IRF5D在PBS（1毫克/公斤/天）皮下用27号针头稀释独自啧/ +和C57BL / 6J小鼠，持续21天。 </li><li>麻醉用异氟烷（5％）的小鼠，并执行一个颈脱位。通过切除切开胸部沿着胸骨的心。提起用钳子心脏，取出心脏。卡扣冻结并存储切除心，直到分析。 </li><li>安装在做卷烟冰冻的心UE持有适合于所用的低温恒温器。削减免疫组化低温恒温器10微米厚的冰冻切片。 </li><li>固定用1％多聚甲醛的磷酸盐缓冲盐水的冷冻切片在室温下20分钟。透用0.5％Triton-X 100的PBS切片5分钟。 注意:多聚甲醛是一种致癌物质监管，需要小心处理。 </li><li>在PBS 200:稀释的抗兔CD64，单核细胞/巨噬细胞标记1。适用于固定部分，并在37℃孵育30分钟。 </li><li>在室温下洗载玻片在PBS中5分钟三次。 </li><li>在PBS 200:稀释的抗大鼠NIMP，伯嗜中性粒细胞标记物1。适用于固定部分，并在37℃孵育30分钟。 </li><li>在室温下洗载玻片5分钟，用PBS三次。 </li><li>稀释二级抗体抗兔Alexa的488缀合和抗大鼠的Alexa 488缀合的1:在PBS 200。 </li><li>应用抗兔Alexa的488缀合和抗大鼠的Alexa在37℃下488偶联的二抗于30分钟的部分相应。抗体的量根据该部分的大小而变化。量应慷慨覆盖部分。在大多数情况下，体积为μL100和200之间。 </li><li>用4&#39;，6-二脒基-2-苯基吲哚（DAPI），为在一个1核染色剂:1,000稀释在PBS中。加入1微升到1毫升的PBS，并在最后一次洗涤步骤使用DAPI。水性安装介质添加到盖玻片，并把它拖到幻灯片。 </li><li>分析通过使用40X油浸物镜聚焦显微镜的荧光标记部分。使用激发波长大于530纳米的488和发射波长为CD64和NIMP。 DAPI与360纳米的激发，在蓝色460纳米发射。通过各自不同的标签区分单核/巨噬细胞和中性粒细胞（N =每抗体10幅图像，各组在3个不同的小鼠）和计数。表达数据作为计数的细胞数。 </LI&gt; </ol> IRF5后抑制细胞6.依赖性舒张<ol><li>麻醉C57BL / 6J对照小鼠和TSK / +小鼠具有和不具有氯胺酮肽治疗（80 - 100毫克/千克）/赛拉嗪（10 - 12.5毫克/千克）的混合物。注射氯胺酮/甲苯噻嗪混合腹腔。手动抑制鼠标。 <ol><li>使用25号或更小针头。针插入腹部的右下象限。注射前撤出柱塞，以确保针不进入血管或肠。 </li></ol></li><li>一旦麻醉足够水平已经达到，确保鼠标在仰卧位，用酒精擦拭浸湿纱布垫皮毛，并用剪刀打开胸腔，取出心脏。 注:放血将安乐死鼠标和通过在脉管释放压力从而减少血管时减少出血使清扫容易。 <ol><li>打开皮肤全光照ガ剪刀，从胸部横向切割到颌骨小心以不干扰下面的组织朝向下颚的前部。 </li><li>通过消除周围的软组织解剖动脉面神经。 </li><li>注意:同时保持在MOPS缓冲液沐浴露组织不要伤害的面神经动脉血管或拖船（2.0毫米氯化钙2·2H 2 O，0.02毫摩尔EDTA，5.0毫米的葡萄糖，4.7毫米氯化钾，1.17毫米硫酸镁4·7H 2 0，3.0毫米MOPS，145毫米氯化钠，1.2毫米和 NaH 2 PO 4·H 2 O，2.0毫米丙酮酸），以防止干燥。一个双目立体变焦解剖显微镜（3.5X的倍率45X）和光纤光源下进行。 </li></ol></li><li>隔离动脉面神经。 <ol><li>掌握面神经近端第一分叉和远端到动脉将被削减。切断从父动脉，颈外动脉的动脉。取出arterie面神经期从对照小鼠，与无IRF5D治疗啧/ +小鼠。 </li><li>同时仍保持在镊子的动脉，切断两个分支脱落面神经，使容器内的被轻轻地把而任何未切割的周围组织可以识别并切断。 </li><li>直到切割女儿都动脉释放血管分叉处继续这种方式。没有必要对之前从取出心脏在脉管切削由于安心压力夹紧血管。把容器在MOPS缓冲液，而另一面神经动脉被解剖并分离。 </li><li> 175微米 - 用125终端直径把它们绑到玻璃吸管导管插入血管。预紧的玻璃吸管和缓冲病险水库MOPS缓冲。 注意:从移液器防止形成气泡。 </li><li>扎到血管用眼科缝线缝合的移液器。 </li></ol></li><li>安装在一个倒triocular MICR容器室用摄像机附着的Oscope。运行通过视频卡尺测量系统的视频在血管屏幕测量。 20 <ol><li>加压它们在两个步骤的过程。首先，在容器等于20毫米汞柱压力之上的高度的MOPS的填充水库，手动打开在储线到容器中的活塞阀。检查周围的关系上下船的长度泄漏的迹象容器中。第二，提高水库60毫米汞柱和复验的容器。 </li><li>一旦在60毫米汞柱压力，使容器中平衡30分钟。 </li></ol></li><li>为了响应于评估血管舒张对乙酰胆碱（10 -7至10 -4 M），preconstrict容器到一个直径50 - 3.0×10 -8至10 -7 M U46619 -使用1其最大直径的75％。允许后该容器，以稳定为6分钟，在2分钟的持续时间的步骤中添加乙酰胆碱浴，使得最终浓度在浴是10 -7，10 -6，10 -5，10 -4。测量在所有三个组中的血管舒张。 </li></ol> 7.隔离心脏细胞外基质的<ol><li>麻醉C57BL / 6J对照小鼠和TSK / +小鼠有和没有用异氟烷（5％）的肽治疗，并执行一个颈脱位。通过切除切开胸部沿着胸骨的心。提起用钳子心脏，取出心脏。 </li><li>把除去心中到烧杯中，并添加15毫升高渗1％十二烷基硫酸钠（SDS）。搅动18小时的1％SDS溶液的心在室温下加入搅拌棒的1％SDS溶液，并把烧杯上的轰动板，打破了细胞。 注意:请注意，如果离开太久的高渗溶液胞外基质会瓦解。相反，如果心中没有搅动足够长的时间在高渗溶液，然后将细胞将不能正确溶解并且会有残留在烧杯细胞碎片。 </li><li>洗在15毫升0.5％的Triton X-100的30分钟。然后洗用15毫升的PBS 15分钟。单独Decellularize每个心脏。 </li><li>卡扣冷冻在液氮中的细胞外基质和powderize用预冷却的研钵和杵将冷冻的心脏细胞外基质。 </li><li>使悬浮液在含有1％抗生素的PBS粉碎基质。在大多数情况下，加入的量是300微升。超声处理30暂停 - 在高设置上冰60秒。 注意:这是非常重要的是，样品保持冷。注意，该悬浮液是非常粘稠由于高蛋白质聚糖含量。 </li><li>确定使用Bradford测定蛋白质浓度。 注:确保有在悬浮抗生物和抗真菌。青霉素/链霉素是最常用的，并建议。 </li></ol> 8.评估核移位通过ImmunohistochemistRY <ol><li>涂覆菜肴和滑动用20微克/毫升在PBS中的心脏细胞外基质的终浓度。吸取500μL心脏细胞外基质的解决方案为100毫米的菜涂层和最终的细胞外基质的解决方案到幻灯片的150微升。 </li><li>通过在50微克/毫升浓度添加IRF5D 24小时培养中，以大鼠新生心肌细胞抑制IRF5。离开控制心肌细胞培养治疗。 </li><li>从细胞质到细胞核用免疫荧光评估IRF5的贩运和共聚焦显微镜分析。识别环保标签的IRF5，确定蓝色DAPI染色的核</li><li>应用的主要抗IRF5抗体。 1:200稀释在PBS中:初级抗体进行1使用。温育30分钟的幻灯片在37℃，并按照与3个5分钟的洗涤步骤。二级抗体是Alexa的488标记的山羊抗小鼠IgG抗体。 </li><li>稀释二级抗体1:1000二lution于PBS中。孵育二级抗体进行30分钟，在37℃。实现与DAPI核染色。在PBS 1000:稀释DAPI 1。洗载玻片共3次，每次5分钟，并添加DAPI到第三和最后的洗涤步骤。 </li><li>通过使用共聚焦显微镜并在细胞核测量荧光强度和细胞溶质如先前捕获贩卖描述10。识别环保标签的IRF5，确定蓝色DAPI染色的细胞核。表现出与它们绿色标记蓝色细胞核细胞含有活化IRF5，它从细胞质至细胞核投放。当IRF5未激活绿色标记在胞质溶胶中找到。 </li><li>分析荧光标记的细胞通过使用40X油浸物镜聚焦显微镜。使用的用于IRF5大于530纳米488纳米波长和发射波长的激发波长。使用360nm的激发波长和460nm的为DAPI的可视化的发光波长。 </li></ol><ol><li>内部和群体之间的重复测量进行方差分析的分析。按照ANOVA与学生t检验邦弗朗尼的修改。表达数据以平均值的标准误差。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal.Biochem. 72 (1-2), 248-254 (1976).</li><li>Bauer, 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=Compensatory+phosphorylation+and+protein-protein+interactions+revealed+by+loss+of+function+and+gain+of+function+mutants+of+multiple+serine+phosphorylation+sites+in+endothelial+nitric-oxide+synthase.">Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of multiple serine phosphorylation sites in endothelial nitric-oxide synthase.</a> J.Biol.Chem. 278 (17), 14841-14849 (2003).</li><li>Weihrauch, D., 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=An+IRF5+Decoy+Peptide+Reduces+Myocardial+Inflammation+and+Fibrosis+and+Improves+Endothelial+Cell+Function+in+Tight-Skin+Mice.">An IRF5 Decoy Peptide Reduces Myocardial Inflammation and Fibrosis and Improves Endothelial Cell Function in Tight-Skin Mice.</a> PLoS One. 11 (4), 0151999 (2016).</li><li>Hoogenboom, H. R., 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=Antibody+phage+display+technology+and+its+applications.">Antibody phage display technology and its applications.</a> Immunotechnology. 4 (1), 1-20 (1998).</li><li>Roehm, N. W., Rodgers, G. H., Hatfield, S. M., Glasebrook, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+colorimetric+assay+for+cell+proliferation+and+viability+utilizing+the+tetrazolium+salt+XTT.">An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT.</a> J Immunol Methods. 142 (2), 257-265 (1991).</li><li>Van Tonder, A., Joubert, A. M., Cromarty, A. 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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=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat Med. 14 (2), 213-221 (2008).</li><li>Ou, 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=L-4F,+an+apolipoprotein+A-1+mimetic,+dramatically+improves+vasodilation+in+hypercholesterolemia+and+sickle+cell+disease.">L-4F, an apolipoprotein A-1 mimetic, dramatically improves vasodilation in hypercholesterolemia and sickle cell disease.</a> Circulation. 107 (18), 2337-2341 (2003).</li><li>Weihrauch, D., 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=Effects+of+D-4F+on+vasodilation,+oxidative+stress,+angiostatin,+myocardial+inflammation,+and+angiogenic+potential+in+tight-skin+mice.">Effects of D-4F on vasodilation, oxidative stress, angiostatin, myocardial inflammation, and angiogenic potential in tight-skin mice.</a> Am J Physiol Heart Circ Physiol. 293 (3), 1432-1441 (2007).</li><li>Roy, S., P, P., Mavragani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRF-5+-+A+New+Link+to+Autoimmune+Diseases.">IRF-5 - A New Link to Autoimmune Diseases.</a> Autoimmune Disorders - Pathogenetic Aspects. , 35 (2011).</li></ol>

The authors declare that they have no competing financial interests.

的目标是设计一个IRF5抑制剂阐明IRF5的炎症，纤维化和血管功能在啧/ +小鼠的心脏的作用。调查结果是IRF5D不诱导增殖或凋亡。此外，炎症减少和血管功能的改善。这些数据表明，IRF5在炎症和纤维化的啧/ +小鼠的心脏的发展的重要机械作用，它具有作为治疗靶的潜力。 第一步是设计的抑制剂。当肽被设计，有几点必须考虑到:序列长度，二级结构的残基容易发生氧化，氨基酸?...

<html>细胞生长和细胞死亡的免疫应答的调节是至关重要的转录因子家族的干扰素调节因子的作用。 IRF5被高亮显示为用于免疫反应类型1之间的调控，炎症促进反应和2型，免疫应答靶向组织修复的关键。 IRF5是在癌症1，和自身免疫2，3，4，5键。 紧皮肤小鼠（TSK / +）为组织纤维化和硬皮病一个模型由于在原纤蛋白-1基因的复制突变。此突变导致一个紧皮肤和增加结缔组织。这些小鼠发展心肌炎症，纤维化和最后心脏衰竭5，6，7，&gt; 8,9。硬皮病是影响美国6约有15万名患者的自身免疫性疾病纤维化。这种疾病的特点是内部器官包括心脏7，8，9，10，11的纤维化。 该研究的天性，需要抑制肽的设计。被选择了该软件的方法利用噬菌体展示传统的方法。的软件的方法是更容易和耗时更少。该RCSB数据库是用来确定适当的结合位点12。研究新设计的肽与重组蛋白的相互作用和集中的结合参数，使用一种技术，称为生物层干涉测量法。生物层干涉测量是基于生物传感器techniqUE决定使用生物传感器和具有约束力的样本结合亲和力，关联和解除关联。该传感器可以是荧光，luminescently，辐射测量和比色标记。测量是基于质量增加或耗竭类似关联和解除关联13,14。本研究的目的是要了解IRF5的心肌炎症和纤维化中的作用。目标是洞察IRF5的在组织纤维化和硬皮病的发展中的作用。

<table><tbody><tr><td>&amp;nbsp;Triton X 100</td><td>Sigma Aldrich</td><td>X100- 100ml</td><td></td></tr><tr><td>Alexa 488-labeled goat anti-mouse IgG antibody&amp;nbsp;</td><td>Thermo Fisher</td><td>A11001</td><td></td></tr><tr><td>Bardford reagent</td><td>Thermo Fisher</td><td>23200</td><td>Pierce&amp;nbsp;</td></tr><tr><td>Biosensors</td><td>Forte-Bio</td><td>MR18-0009</td><td></td></tr><tr><td>CD64 (H-250)</td><td>Santa Cruz Biotechnologies</td><td>sc-15364</td><td></td></tr><tr><td>CellEvent Caspase-3/7 Substrate</td><td>Thermo Fisher/Life Technologies</td><td>C10427</td><td></td></tr><tr><td>CellTiter AQueous One Solution Cell Proliferation Assay kit</td><td>Promega</td><td>G3580</td><td>Promega</td></tr><tr><td>DAPI (4&amp;prime;,6-diamidino-2-phenylindole)</td><td>Thermo Fisher</td><td>D-1306</td><td>1:1,000 dilution in PBS</td></tr><tr><td>donkey anti rat Alexa 488</td><td>Thermo Fisher</td><td>A-21208</td><td>1:1,000 dilution in PBS</td></tr><tr><td>ECL plus</td><td>GE healthcare/Amersham</td><td>RPN2133</td><td>After a lot of trial and error we came back to this one</td></tr><tr><td>Eclipse TE 200-U microscope with EZ C1 laser scanning software</td><td>Nikon</td><td></td><td></td></tr><tr><td>goat anti rabbit Alexa 488</td><td>Thermo Fisher</td><td>A-11008</td><td>1:1,000 dilution in PBS</td></tr><tr><td>HRP&amp;nbsp; anti-goat</td><td>Santa Cruz Biotechnologies</td><td>sc-516086</td><td>!:10,000 dilution in TBS</td></tr><tr><td>HRP donkey anti-mouse</td><td>Santa Cruz Biotechnologies</td><td>sc-2315</td><td>1:10,000 dilution in TBS</td></tr><tr><td>ICAM-1 antibody</td><td>Santa Cruz Biotechnologies</td><td>sc-1511</td><td>1:200 dilution in PBS</td></tr><tr><td>IRF5 antibody (H56)</td><td>Santa Cruz Biotechnologies</td><td>sc-98651</td><td></td></tr><tr><td>Micro plate reader Elx800</td><td>Biotek</td><td></td><td></td></tr><tr><td>NIMP neutrophil marker</td><td>Santa Cruz Biotechnologies</td><td>sc-133821</td><td>1:200 dilution in PBS</td></tr><tr><td>Octet RED</td><td>Forte Bio</td><td></td><td>protein-protein binding</td></tr><tr><td>Peptide design&amp;nbsp; Medit SA software</td><td>RCSB.org</td><td></td><td></td></tr><tr><td>Recombinant IRF5 protein synthesis</td><td>TopGene Technologies</td><td></td><td>protein expression, synthesis service</td></tr><tr><td>sodium dodecyl phosphate</td><td>Sigma Aldrich</td><td>436143</td><td>detergent</td></tr><tr><td>Ketamine</td><td>Pharmacy</td><td></td><td>Schedule III controlled substance, presciption required&amp;nbsp;</td></tr><tr><td>Xylazine</td><td>MedVet</td><td></td><td></td></tr><tr><td>3.5X-45X Trinocular Dissecting Zoom Stereo Microscope with Gooseneck LED Lights</td><td>Am Scope</td><td>SKU: SM-1TSX-L6W</td><td></td></tr><tr><td>Zeba Desalting Columns</td><td>Thermofisher</td><td>2161515</td><td></td></tr><tr><td>Endothelial Basal Media EBM Bullet kit</td><td>Lonza</td><td>CC-3124</td><td>kit contains growth supplemets</td></tr><tr><td>VIA-100K</td><td>&amp;nbsp;Boeckeler Instruments</td><td></td><td></td></tr><tr><td>4 - 15% TGX gel</td><td>Bio-Rad</td><td>5671081</td><td></td></tr><tr><td>MedSuMo software</td><td>Medit, Palaiseau, France</td><td></td><td></td></tr><tr><td>Laemmli Buffer</td><td>BioRad</td><td></td><td></td></tr></tbody></table>

vasodilation of isolated vessels and the isolation of the extracellular matrix of tight-skin mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

在图1中展示了结果表明如何设计的肽。 图1，左上，示出了由多个激酶磷酸化在IRF5的区域（2黄色箭头之间，氨基酸（AA）425-436）。 图1，右上，显示了一个黄色椭圆形，其中IRF5的磷酸化结构域结合。 3DSH的二聚结构被旋转以观察一个裂口或谷到螺旋2（aa303-312）的左侧。这是IRF5的磷酸尾域假定当它被完全激活（ 即 ，丝氨酸磷?...

Watch this Scientific Journal Video about Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice at JoVE.com

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

隔离容器的血管扩张和细胞外基质的紧皮肤小鼠隔离

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

vasodilation-isolated-vessels-isolation-extracellular-matrix-tight

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.0K 次观看.  Medical College of Wisconsin. We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide. 

视频: Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

科研

发育生物学

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

生物工程

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

在背皮褶商会鼠标的缺血性组织损伤:一个皮瓣模型，探讨急性缺血持续

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

医学

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

在鼠耳皮肤的免疫反应的活体多光子成像诱导缺血再灌注损伤

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

从鼠标阻力动脉分离微血管内皮管

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

生物学

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

划痕迁移分析和背皮折叠室的体外和在Vivo分析伤口愈合

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

在人类内皮周细胞的相互作用使用基质凝胶插件检测小鼠的体内研究

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

用迈尔斯测定法评价皮肤血管 Hyperpermeability 诱导剂的含量

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow increases with decreasing diameter from arteries to arterioles that ultimately feed into capillaries. Due to their smaller size and location in the parenchyma, arterioles have been relatively understudied and with less reproducibility in findings than surface pial arteries. Regardless, arteriolar endothelial cell structure and function&#8212;integral to the physiology and etiology of chronic degenerative diseases&#8212;requires extensive investigation. In particular, emerging evidence demonstrates that compromised endothelial function precedes and exacerbates cognitive impairment and dementia.
In the parenchymal microcirculation, endothelial K+ channel function is the most robust stimulus to finely control the spread of vasodilation to promote increases in blood flow to areas of neuronal activity. This paper illustrates a refined method for freshly isolating intact and electrically coupled endothelial &#34;tubes&#34; (diameter, ~25 &#181;m) from mouse brain parenchymal arterioles. Arteriolar endothelial tubes are secured during physiological conditions (37 &#176;C, pH 7.4) to resolve experimental variables that encompass K+ channel function and their regulation, including intracellular Ca2+ dynamics, changes in membrane potential, and membrane lipid regulation. A distinct technical advantage versus arterial endothelium is the enhanced morphological resolution of cell and organelle (e.g., mitochondria) dimensions, which expands the usefulness of this technique. Healthy cerebral perfusion throughout life entails robust endothelial function in parenchymal arterioles, directly linking blood flow to the fueling of neuronal and glial activity throughout precise anatomical regions of the brain. Thus, it is expected that this method will significantly advance the general knowledge of vascular physiology and neuroscience concerning the healthy and diseased brain.

Intensive preparation of intact mouse cerebral endothelial &#34;tubes&#34; from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca2+] and membrane potential.

小鼠脑实质小动脉内皮的分离及功能分析

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow ...