资金来源/编号分子生物学失血性休克GM053789

1。仪器及手术现场准备： 所有的实验程序进行使用无菌操作技术。实验区开始之前，必须彻底清洗和消毒。台式，应进行消毒，空气干燥，然后用70％的酒精抹允许。将一个蓝色的手术垫和无菌区，敷在试点工作区。 所有材料和工具，高压灭菌消毒后方可使用。收到无菌注射器和针头。研究者应适当garbed白大褂，口罩，无菌手术手套。 我们的手术器械，高压灭菌消毒每天晚上。他们手术后，使用抗菌肥皂和水清洗。他们被允许在一个干净的手术蓝色垫干。他们然后小心地放入灭菌袋和消毒，以备后用。 打开热珠灭菌，以确保它达到适当的温度 - ° F在开始实验前300-350。这种灭菌器将被用来清洁之间在各自的小鼠实验程序的工具。获取一个不锈钢仪器托盘，并填写1 / 3，用70％乙醇的方式。应该有足够的70％的乙醇，以支付您的手术器械。打开循环加热垫，并放置一个手术蓝色垫在它和无菌敷料领域最重要的是。这种热水器是使用鼠标有足够的温暖，以确保在实验过程和恢复期。放置在无菌的战地包扎消毒纱布，注射器，针头和1.5ml的离心管。开放任何消毒和其他物品时要小心，不要去触摸污染。最好是这样做安装程序时，使用无菌手套。 2。 Pseudofracture模型 - 实验步骤： 1。麻醉诱导和鼠标的定位。 从管理的Pentobarbitol腹腔注射（70mg/kg）。这是通过先解除从笼子里的鼠标，它的尾巴的近端。鼠标然后必须称重，计算anaesthesic正确的剂量。接下来，放在笼子顶部的动物，同时仍持有它的尾巴。鼠标，仅仅落后的前爪两侧用大拇指和中指抓住鼠标的脖子颈背。食指是用来拉背部的皮肤上的头部/颈部区域和部分固定头部。而无名指上按下鼠标的脊椎腰椎地区，​​老鼠的尾巴是小指和无名指之间举行。麻醉约5分钟内应采取充分的效果，应与反射试验证实。 后的动物都睡着了，在仰卧位放置鼠标到一个有机玻璃板，并使用一个松散的循环磁带技术固定用胶带其四肢。宽松的循环技术，只需要切细条状的磁带，磁带环绕着每个下肢的远端部分松散。磁带，然后停留本身超过磁带的左侧连接到电路板。这使得要一致的方式，以及确保对小鼠的四肢进行实验操作，假设一个更自然的位置。动物的腹部，腹股沟和大腿的地区，然后使用我们的奥斯特A5的快船队（40刀片），以帮助保持无菌领域剃光。一个4x4纱布浇上用优碘消毒手术区，然后消灭。一个无菌战地包扎，然后折叠，并通过鼠标搭着只有较低的四肢和头部，让曝光。 固定和灭菌后，将与异氟醚鼻锥几秒钟之前开始的实验过程中对老鼠的鼻子。鼻锥由一个50CC的锥形管，用纱布填充。管底的一半是创建一个老鼠的鼻子放进空间。被放在锥形切割结束，以确保异氟醚蒸气管在不使用时保持一个上限（一个组织的存储容器，即直边宽口瓶底部）。应添加1毫升的异氟醚的纱布内锥体。一丝不苟应支付给鼠标使用麻醉过程中，特别是使用异氟醚通过鼻锥。一旦动物的呼吸开始放慢，就可以开始实验，并应拆除和关闭的鼻锥。 如果需要额外的麻醉是整个实验的任何部分，额外的苯巴比妥可能被注射或异氟醚管理。 isofluorane管理的替代方法和指引建议的更新结束一个精确的蒸发器的使用，以便更好地控制麻醉，如果可行的话。但是，必须密切关注鼠标的呼吸生理学与这些方法中的任何保证。 2。 Pseudofracture实验。 伪骨折是一种结合软组织损伤，其次是注射到受伤的肌肉骨骼的解决方案，并进行双边的后肢。任何收件人的小鼠实验操作开始之前，必须准备骨解决方案。 3。骨溶液的制备。 骨的解决方案，准备从一个捐助国鼠标将足以供3受体小鼠。应该使用的是年龄和体重相匹配的收件人实验小鼠同源捐助鼠标。 捐赠者将鼠​​标安乐死与吸入异氟醚。捐助者将被贴在有机玻璃板与认真注意的磁带，只在脚的一角下肢。下肢必须仔细剃光优碘在彻底覆盖，然后用酒精为不育消灭。然后将股骨和胫骨手术切除在无菌的方式从双下肢。为了消除下肢长骨，使之成为在腹股沟区域的皮肤外科手术切口，并继续切开皮肤，沿下肢的长度到脚踝。收回的皮肤，从皮下筋膜上最大的曝光率的内侧和外侧的肌肉解剖。在于前和外侧胫骨（胫前肌，趾长伸肌，伸指hallicus长肌，腓骨长肌，腓骨短）和滑动刀片近端和远端解除肌肉干净远离的肌肉，下插入一个剪刀的刀片底层骨。胫骨后内侧底面重复这种技术单独下方的胫骨（腓肠肌，比目鱼肌，跖，胫骨后，屈趾长伸肌，屈肌hallicus长肌）的肌肉。应使用一个最小的额外消耗后，在滑动远端时，以单元距胫骨，腓骨。在这些腿部肌肉的近端切筋接近其插入尽可能收回远端的肌肉，而肌肉群拉向下时尚的小拖船将释放这些肌肉从踝关节的远端附件。踝关节可以直接用剪刀划破。不要脱离在这一点上的股骨胫骨！这种连接提供了额外的杠杆作用，将有助于从髋关节股骨解剖。按照从股骨的肌肉释放的一个类似的技术和执行此向后内侧和外侧如果需要的话。削减这些肌肉群的远端附件释放他们从膝关节。继续剥离，并遵循下面的肌肉解剖髋关节股骨结束。为了帮助剥离，皮肤切口应尽可能要求，以达到充分暴露延长。股骨髋关节的运动，可以帮助找到在剥离过程中的股骨近端。 一旦股骨与胫骨附着删除捐助鼠标放在无菌4x4gauze。这两块骨头，可在膝关节分开，通过简单地在一个单独的手抓住每个骨骼和轻轻扭曲/旋转相反的方向沿其长轴的两块骨头。温和的清扫和处理这两块骨头是建议，因为他们可以很容易地与任何手工操作过度劳累断裂。要删除其余的肌腱附件，长骨，用消毒干片的4x4的纱布缠骨的长度。纱布紧紧抓住对骨把它全长骨几次，这将关闭骨面刮，剩余的组织，因为他们将坚持以纱布，并迅速和容易被拉从骨子里。收集的骨头应该放在直接进入无菌的1.5ml的离心管放在冰上运输。 这四个捐助者的骨骼，将采取生物安全柜骨解决方案准备。油烟机在使用前必须彻底消毒，置于无菌敷料在工作区域内罩的领域。将无菌迫击炮和杵，无菌8mL管和无菌磷酸盐缓冲液（PBS）在工作区。一个1ML pipetteman，和各自的技巧应可罩内。 一个无菌镊子对应当用于去除，在无菌的方式收获骨骼1.5ml的离心管放入砂浆;杵将被用来轻轻破碎的骨头。磷酸化吃的缓冲液是用于重悬注射碎骨的车辆。移液器1ML到砂浆的PBS继续粉碎额外的圆周运动，其余的碎片，以确保充分的再悬浮。然后添加另一个的PBS液1ml，继续粉碎创建的“骨解决方案”。该解决方案应该有一个粉红色的色调，和会有的，不能完全打破了迫击炮的底部留下的残余。缓慢倒入砂浆的解决方案到8mL管，捕获的最大音量，同时确保较大的残余仍然落后于砂浆。此骨的解决方案应保持在冰上，直到其实验使用，将被转移到注射液1ml注射器。一个20个针头稍后会连接到1ml注射器，含骨的解决方案，以便来管理它的鼠标。这种大型的针头大小的选择，以确保所有的碎骨后注射进入该地区并没有阻止针。 注射前，骨的解决方案“下拉到收件人鼠标放在一个文化麦康凯琼脂平板（2448小时孵化）。这是骨解决方案，以确保无菌。骨溶液的pH值也必须检查，以确保一个中性的pH值。 4。软组织损伤。 这种软组织损伤，是一种双边的后肢挤压伤。鼠标的初步定位与后肢轻微的绑架和横向旋转，在方便正确的肌肉群，膝屈肌（股二头肌，半腱肌和半膜肌的肌肉）帮助。一个大18厘米止血，将被用于执行挤压伤。这些应用止血的力量分布进行了分析，发现是270psi使用Topaq压力分析系统由传感器产品公司的止血钳，必须围绕大腿后侧肌肉，中点钳位沿股骨与它的凸曲线面临的股骨。止血应被锁定，只在第一次点击关闭，并保持30秒。仔细注意这种止血钳的位置是非常重要的 - 它不能被夹在股骨，以确保它不会断裂。这是应以一致的方式进行，每次保证小鼠之间的重复性伤害。这双下肢挤压伤。 5。骨的解决方案注射。 收件人实验将鼠标麻醉，准备如前所述，将发生软组织损伤，这骨注射前。 骨的解决方案将被注入收件人鼠标双边粉碎大腿肌肉。使用20号针头，0.15mL这个解决方案将被注入到每个大腿的后部肌肉。透过皮肤进入针约2〜3mm时，注意插入针的距离 - 你会觉得斜角的提示，只是对股骨接触，这一立场是非常理想的。注入骨解决方案。撤回的针头和注射部位迅速放在无菌戴手套的手指停止任何伤口的骨液回流。保持这个手指有几秒钟。 然后将鼠标放回入笼，并允许直接作为麻醉消退运动的充分自由。必须设置适当的疼痛管理，鼠标唤醒。 6。 Post-operative/recovery时期。 宽松循环磁带被删除，动物被放置到一个干净的笼子里，这是保存在几个小时后恢复循环加热垫。一个额外的热量灯，如果需要，应确保适当的温暖。 食物和水会随时可用。 必须管理的动物开始从麻醉中唤醒，以正确的管理疼痛的镇痛。丁丙诺啡（为0.1mg/kg）皮下注射动物开始的体力活动，但不前，所以不会危及呼吸功能。 小鼠应不断监测在麻醉过程中，直到恢复从麻醉中或实验端点。小鼠应非常仔细地监测在术后期间，需要任何额外的止痛药，应根据需要管理。监控动物的身体活动，呼吸状态，食物和水的摄入量和窘迫的任何迹象（吃力的呼吸，疼痛，饮食习惯的改变），应处理相应。 您的手术器械放入70％酒精，并用无菌纱布，然后放入微珠〜20秒动物之间的消毒灭菌。取出手术器械，并用70％酒精喷洒，以帮助他们冷静下来。放置到STerile战地包扎。确保有左无酒精的文书，将滴灌进入下一个动物。 3。比较双侧股骨骨折模型 - 实验步骤： 实验小鼠，将最初的麻醉和准备。周围约2 - 3mm的上述后肢膝关节凸面临膝关节表面应夹住一个大18厘米止血。上方止血和臀部夹紧止血与你的食指放在你的拇指。务必感到扭曲，使你知道你正在打破之前的股骨。然后捻止血钳逆时针扭另一只手顺时针的同时。 与对侧腿重复的过程。 4。多重创伤模型 - Pseudofracture失血性休克的组合实验程序： 收件人将鼠标最初的麻醉和准备。接受结合伪骨折失血性休克的小鼠，将之前pseudofracture的过程中有股动脉插管，然后将导管从这个hemorrhaged伪骨折后已经完成。 5。成功的秘密： 一般： <ul><li>请确保您采取额外的努力，保持尽可能无菌。 </li><li>一丝不苟应支付给鼠标的整体生理状况，包括呼吸频率，在施行麻醉，特别是通过鼻锥的异氟醚。 </li></ul>骨溶液的制备： <ul><li>作为温和操纵的骨头 - 可以很容易断裂与过度劳累，在简单的手工操作！ </li><li>应确保从胫骨腓骨搬迁后清扫。 </li><li>首先，在第一破碎的骨头轻轻 - 砂浆，以便保持在所有的片段！ </li><li>最初只添加1毫升PBS，继续粉碎剩余的碎骨，一旦已经形成了良好的悬挂，加入剩余的1ML。 </li><li>只有从解决迫击炮和杵应收集 - 没有较大的剩余的组织，不能完全分解 - 这些是块/得到投诉后，注射针进入收件人鼠标。 </li></ul>软组织损伤： <ul><li>这种止血钳小心堆放重要的是 - 不超过股骨或将断裂。止血钳在正确的肌肉（大腿后侧 - 膝关节屈肌）重复性损伤。 </li></ul>骨的解决方案注射： <ul><li>注入解决方案时，要注意插入针的距离 - 你会觉得斜角的提示，只是对股骨接触，这一立场是非常理想的。 </li><li>撤回针头和注射部位迅速放在手指停止任何伤口的骨液回流。将手指有几秒钟。 </li></ul> 6。发布执行的担忧： <ul><li>检查的腿部感染。 </li><li>检查骨髓液培养。 </li><li>动物可能无法使用后肢作为实验操作及相关炎症。管理适当的疼痛。 </li></ul>

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U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deterioration+of+the+immune+system+after+trauma:+signals+and+cellular+mechanisms.">Deterioration of the immune system after trauma: signals and cellular mechanisms.</a> Inn. Immun. 14, 333-344 (2008).</li><li>Maier, B., LeFering, R., Lhenert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+versus+late+onset+of+multiple+organ+failure+is+associated+with+differing+patterns+of+plasma+cytokine+biomarker+expression+and+outcome+after+severe+trauma.">Early versus late onset of multiple organ failure is associated with differing patterns of plasma cytokine biomarker expression and outcome after severe trauma.</a> Shock. 28, 668-674 (2007).</li><li>Mestas, J., Hughes, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+not+men:+differences+between+mouse+and+human+immunology.">Of mice not men: differences between mouse and human immunology.</a> J. Immunol. 172, 2731-2738 (2004).</li><li>Hoth, J. J., Wells, J. D., Brownlee, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll+like+receptor+4-dependent+responses+to+lung+injury+in+a+murine+model+of+pulmonary+contusion.">Toll like receptor 4-dependent responses to lung injury in a murine model of pulmonary contusion.</a> Shock. 31, 376-381 (2009).</li><li>Matsutani, T., Samy, A. n. a. n. t. h. a., Rue, T. S., W, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+prolactin-/-+mice:+effect+of+trauma-hemorrhage+on+splenocyte+functions.">Transgenic prolactin-/- mice: effect of trauma-hemorrhage on splenocyte functions.</a> Am J Physiol Cell Physiol. 288, 1109-1116 (2005).</li><li>Matsutani, T., Samy, A. n. a. n. t. h. a., Kang, T. S., S-C, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+genetic+background+influences+severity+of+immune+responses+following+trauma-hemorrhage.">Mouse genetic background influences severity of immune responses following trauma-hemorrhage.</a> Cytokine. 30, 168-117 (2005).</li><li>Tsukamoto, T., Pape, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+trauma+research:+What+are+the+options.">Animal models for trauma research: What are the options.</a> Shock. 31, 3-10 (2008).</li><li>DeMaria, E. J., Pellicane, J. V., Lee, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemorrhagic+shock+in+endotoxin+resistant+mice+:+Improved+survival+unrelated+to+deficient+production+of+tumor+necrosis+factor.">Hemorrhagic shock in endotoxin resistant mice : Improved survival unrelated to deficient production of tumor necrosis factor.</a> J Trauma. 35, 720-724 (1993).</li><li>Jamsa, T., Jalovaara, P., Peng, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three-point+bending+test+and+peripheral+quantitative+computed+tomography+analysis+in+the+evaluation+of+the+strength+of+mouse+femur.">Comparison of three-point bending test and peripheral quantitative computed tomography analysis in the evaluation of the strength of mouse femur.</a> Bone. 23, 155-161 (1998).</li><li>Bounarens, F., Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+standard+closed+fracture+in+laboratory+animal+bone.">Production of a standard closed fracture in laboratory animal bone.</a> J Ortho Res. 2, 97-101 (1984).</li><li>Holstein, J. H., Menger, M. D., Culemann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+locking+femur+nail+for+mice.">Development of a locking femur nail for mice.</a> J Biomech. 40, 215-219 (2007).</li><li>Holstein, J. H., Garcia, P., Histing, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+establishment+of+defined+mouse+models+for+the+study+of+fracture+healing+and+bone+regeneration.">Advances in the establishment of defined mouse models for the study of fracture healing and bone regeneration.</a> J Orthop Trauma. 23, S31-S38 (2009).</li><li>Histing, T., Garcia, P., Matthys, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+internal+locking+plate+to+study+intramembranous+bone+healing+in+a+mouse+femur+fracture+model.">An internal locking plate to study intramembranous bone healing in a mouse femur fracture model.</a> J Orthop Research. 28, 397-402 .</li><li>Manigrasso, M. B., O'Connor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+closed+femur+fracture+model+in+mice.">Characterization of a closed femur fracture model in mice.</a> J Ortho Trauma. 18, 687-695 (2004).</li><li>Mark, H., Bergholm, J., Nilsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+external+fixation+method+and+device+to+study+fracture+healing+in+rats.">An external fixation method and device to study fracture healing in rats.</a> Acta Orthop Scand. 74, 476-487 (2003).</li><li>Bhandari, M., Shanghnessy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+minimally+invasive+percutaneous+technique+of+intramedullary+nail+insertion+in+an+animal+model+of+fracture+healing.">A minimally invasive percutaneous technique of intramedullary nail insertion in an animal model of fracture healing.</a> Arch Orthop Trauma Surg. 121, 591-593 (2001).</li><li>Sonanis, S. V., Lampard, A. L., Kamat, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+to+remove+a+bent+femoral+intramedullary+nail+and+broken+interlocking+screw.">A simple technique to remove a bent femoral intramedullary nail and broken interlocking screw.</a> J Trauma. 63, 435-438 (2007).</li><li>Kobbe, P., Kaczorwoski, D. J., Vodovotz, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+exposure+of+bone+components+to+injured+soft+tissue+induces+Toll-like+receptor+4-dependent+systemic+inflammation+with+acute+lung+injury.">Local exposure of bone components to injured soft tissue induces Toll-like receptor 4-dependent systemic inflammation with acute lung injury.</a> Shock. 30, 686-691 (2008).</li><li>Bumann, M., Henke, T., Gerngross, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+haemorrhagic+shock+on+fracture+healing.">Influence of haemorrhagic shock on fracture healing.</a> Lang Arch Surg. 388, 331-338 (2003).</li><li>Santry, H. P., Alam, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+resuscitation:+past,+present,+and+the+future.">Fluid resuscitation: past, present, and the future.</a> Shock. 33, 229-241 (2010).</li><li>Hierholzer, C., Billiar, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+in+the+early+phase+of+hemorrhagic+shock.">Molecular mechanisms in the early phase of hemorrhagic shock.</a> Lang Arch Surg. 386, 302-308 (2001).</li><li>Chaudry, I., Ayala, A., Ertel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemorrhage+and+resuscitation:+immunological+aspects.">Hemorrhage and resuscitation: immunological aspects.</a> Am J Physiol. 259, 63-678 (1990).</li><li>Kawasaki, T., Hubbard, W. J., Choudhry, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trauma-hemorrhage+induces+depressed+splenic+dendritic+cell+functions+in+mice.">Trauma-hemorrhage induces depressed splenic dendritic cell functions in mice.</a> J Immunol. 177, 4514-4520 (2006).</li><li>Kohut, L., Darwiche, S. S., Frank, A. M., Brumfield, J. M., Billiar, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixed+volume+or+Fixed+Pressure:+A+Murine+Model+of+Hemorrhgaic+Shock.">Fixed volume or Fixed Pressure: A Murine Model of Hemorrhgaic Shock.</a> J Vis Exp. , (2010).</li><li>Menzel, C. L., Pfeifer, R., Darwiche, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+lower+extremity+damage+in+mice:+time+course+of+organ+damage+and+immune+response.">Models of lower extremity damage in mice: time course of organ damage and immune response.</a> J Surg Res. 166, e149-e156 (2011).</li><li>Pfeifer, R., Kobbe, P., Darwiche, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+hemorrhage+in+the+induction+of+systemic+inflammation+and+remote+organ+damage:+Analysis+of+combined+pseudo-fracture+and+hemorrhagic+shock.">Role of hemorrhage in the induction of systemic inflammation and remote organ damage: Analysis of combined pseudo-fracture and hemorrhagic shock.</a> J Orthop Res. 29, 270-274 (2011).</li></ol>

动物实验机构动物护理和使用委员会和美国匹兹堡大学，AALAS / AAALAC认证认可机构的办公室进行研究和遵守规定的准则和法规的规定执行。动物来源包括杰克逊实验室和查尔斯河实验室。所有的动物进行广泛的健康保证通过每个供应商，以及大学，匹兹堡的内部动物卫生监测方案。进行这项研究是根据美国政府使用脊椎动物校长。该计划是与美国农业部注册，并已与公共卫生实验室动物福利处的保证信。

Pseudofracture，重现的无菌肌肉骨骼损伤的小鼠模型，可以评估创伤后的免疫反应。通过休闲的长骨骨折的特点，pseudofracture模型免疫模仿下肢骨折环境：受伤的肌肉和软组织暴露不破坏原生骨骨和骨髓受损 38,39双相免疫反应，可以看出以下。 pseudofracture创伤早期hyperinflammatory的响应，可以看出在6个小时的延迟免疫抑制的第二个组件描述为大约48小时的低谷到高峰组成。这种模式有助于克服?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Surgical blue pad</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>50-7105</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Field dressings</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>NC9517505</td>
<td>&nbsp;</td>
<tr>
<td>Circulating heating pad 18"x26"</td>
<td>&nbsp;</td>
<td>Harvard</td>
<td>py872-5272</td>
<td>&nbsp;</td>
<tr>
<td>Hot bead instrument sterilizer</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>11156-002</td>
<td>&nbsp;</td>
<tr>
<td>Stainless steel tray 8" x 11"</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>62687-049</td>
<td>&nbsp;</td>
<tr>
<td>Plexiglass boards (10x15x0.5cm) </td>
<td>&nbsp;</td>
<td>University of Pittsburgh Machine shop</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tape rolls 1"</td>
<td>&nbsp;</td>
<td>Corporate Express</td>
<td>MMM26001</td>
<td>&nbsp;</td>
<tr>
<td>50cc conical tube</td>
<td>&nbsp;</td>
<td>can be purchased through any global vendor</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Straight side wide mouth jars (used as cap for nose cone)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>159000-058</td>
<td>&nbsp;</td>
<tr>
<td>Oster A5 clippers w. size 40 blade</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>10749-020</td>
<td>&nbsp;</td>
<tr>
<td>Surgical scissors (straight &#x2013; 12cm)</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14068-12</td>
<td>&nbsp;</td>
<tr>
<td>Hemostats curved -18cm</td>
<td>&nbsp;</td>
<td>Harvard</td>
<td>81331718</td>
<td>&nbsp;</td>
<tr>
<td>Forceps (0.8mm-tip, curved-10cm)</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11050-10</td>
<td>&nbsp;</td>
<tr>
<td>Gauze 4"x4"</td>
<td>&nbsp;</td>
<td>can be purchased through any global vendor</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5cc microfuge tube</td>
<td>&nbsp;</td>
<td>can be purchased through any global vendor</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ice bucket</td>
<td>&nbsp;</td>
<td>can be purchased through any global vendor</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mortar and Pestle </td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-961AA</td>
<td>&nbsp;</td>
<tr>
<td>1cc syringe w/ 25G needle</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>14-826-88</td>
<td>&nbsp;</td>
<tr>
<td>20G needle</td>
<td>&nbsp;</td>
<td>can be purchased through any global vendor</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1mL pipetteman</td>
<td>&nbsp;</td>
<td>can be purchased through any global vendor</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1mL pipette tips</td>
<td>&nbsp;</td>
<td>can be purchased through any global vendor</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Falcon polystyrene 8ml tubes </td>
<td>&nbsp;</td>
<td>VWR</td>
<td>60819-331</td>
<td>&nbsp;</td>
<tr>
<td>Sterilization pouch 3"x8"</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>24008</td>
<td>&nbsp;</td>
<tr>
<td>Sterilization pouch 5"x10"</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>24010</td>
<td>&nbsp;</td>
<tr>
<td>MacConkey II Agar plate</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>221172</td>
<td>&nbsp;</td>
<tr>
<td>Ethyl Alcohol - 200 proof </td>
<td>&nbsp;</td>
<td>Pharmaco-AAPER</td>
<td>&nbsp;</td>
<td>[70%]</td>
</tr>
<tr>
<td>Pentobarbital Sodium (Nembutol Sodium Solution)</td>
<td>&nbsp;</td>
<td>Ovation </td>
<td>&nbsp;</td>
<td>70mg/kg</td>
</tr>
<tr>
<td>Aerrane (Isoflurane)</td>
<td>&nbsp;</td>
<td>Baxter </td>
<td>&nbsp;</td>
<td>99.9%</td>
</tr>
<tr>
<td>Triadine Povidone Iodine (Betadine)</td>
<td>&nbsp;</td>
<td>Triad disposables</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine HCl</td>
<td>&nbsp;</td>
<td>Bedford Laboratories</td>
<td>&nbsp;</td>
<td>0.1mg/kg</td>
</tr>		</tbody>
		</table>
		</div>

pseudofracture: an acute peripheral tissue trauma model

创伤后有一个早期的超反应性的炎症反应，可导致多器官功能障碍和创伤患者的死亡率高;这种反应往往伴随着延迟免疫抑制，增加感染的临床并发症， 还可以增加死亡率1-9。许多研究已开始在免疫系统的反应，以评估这些变化的创伤。 10-15 免疫学研究的大力支持，通过多种转基因和基因敲除小鼠体内建模;这些菌株在详细调查援助，以评估参与免疫反应的分子途径 16-21 在实验小鼠创伤建模的挑战是长期的调查，在小鼠骨折的内固定技术，可以复杂，不容易重现 。22-30 这pseudofracture模式，一个很容易复制的创伤模型，克服了这些困难，免疫模仿下肢骨折的环境，同时允许在动物和长期生存的自由运动，没有持续的，长期使用麻醉。其目的是重新长骨骨折的特点，受伤的肌肉和软组织暴露不破坏原生骨受损的骨骼和骨髓。 pseudofracture模型由两部分组成：一个双边的后肢肌肉挤压伤，其次是注射到这些受伤的肌肉骨解决方案，。准备收获从一个年龄和体重相匹配的同源捐助两个后肢长骨骨的解决方案。然后，这些骨头粉碎，重悬于磷酸盐缓冲生理盐水创建骨解决方案。 双侧股骨骨折是四肢创伤中常用的和行之有效的模式，并pseudofracture模型的发展过程中比较模型。各种可用的骨折模型当中，我们选择使用我们的pseudofracture比较软组织损伤的骨折闭合方法，因为我们想无菌但周围组织的比例严重创伤模型。 31 失血性休克是一种严重创伤的设置共同发现，和全球的低灌注创伤模型增加了一个非常相关的元素。32-36的pseudofracture模型可以很容易地与失血性休克模型为多发性创伤的严重程度高的模型相结合37。

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Pseudofracture: An Acute Peripheral Tissue Trauma Model

Pseudofracture，重现的无菌肌肉骨骼损伤的小鼠模型，可以为后期的长期创伤后的免疫反应的评价。本文介绍的模式一步一步的程序执行，包括允许多发性创伤的研究实验模型组合的潜力。

Pseudofracture：急性周围组织的创伤模型

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; ...

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Medicine

14.6K 次观看.  University of Pittsburgh. Pseudofracture，重现的无菌肌肉骨骼损伤的小鼠模型，可以为后期的长期创伤后的免疫反应的评价。本文介绍的模式一步一步的程序执行，包括允许多发性创伤的研究实验模型组合的潜力。

视频: Pseudofracture: An Acute Peripheral Tissue Trauma Model

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

可视化和肝脏微循环血流量和耗氧量分析：急性肝炎模型中的应用

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

科研

医学

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

急性脑外伤小鼠其次是纵向双光子成像

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

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 ...

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

异位骨化直接鼠标创伤/烧伤模型

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

A Saline/Bipolar Radiofrequency Energy Device As an Adjunct for Hemostasis in Solid Organ Injury/Trauma

Solid organ (liver, spleen, and kidney) hemorrhage is often life-threatening and can be difficult to stop in critically ill patients. Traditional techniques for arresting this ongoing bleeding include coagulation by high voltage electrocautery, topical hemostatic application, and the delivery of ignited argon gas. The goal of this study/video was to demonstrate the efficacy of a new energy device for arresting persistent solid organ hemorrhage.A novel instrument utilizing bipolar radiofrequency (RF) energy which acts to ignite/boil dripping saline from a simple handpiece is employed to arrest ongoing bleeding from solid organ injuries in a porcine model. This instrument is extrapolated from experience within elective hepatic resections. An escalating series of injuries to solid organs within a porcine model will be created. This will be followed by arresting hemorrhage with this novel energy device in sequence. A standard suction device will also be employed. This simple saline/RF energy instrument has the potential to arrest ongoing solid organ surface/capsular bleeding, as well as moderate hemorrhage associated with deep lacerations.

The goal of this publication is to demonstrate the potential application of a novel device using simulated solid organ injuries in a porcine model.

盐水/双极射频能量装置作为固体器官损伤/创伤中血侧体

Solid organ (liver, spleen, and kidney) hemorrhage is often life-threatening and can be difficult to stop in critically ill patients. Traditional ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

骨关节炎模型中骨髓、骨髓、淋巴结和合成组织内免疫细胞子集的流动细胞测量分析

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Halogenated Agent Delivery in Porcine Model of Acute Respiratory Distress Syndrome via an Intensive Care Unit Type Device

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent need to find effective therapies. Preclinical studies have shown that inhaled halogenated agents may have beneficial effects in animal models of ARDS. The development of new devices to administer halogenated agents using modern intensive care unit (ICU) ventilators has significantly simplified the dispensing of halogenated agents to ICU patients. Because previous experimental and clinical research suggested potential benefits of halogenated volatiles, such as sevoflurane or isoflurane, for lung alveolar epithelial injury and inflammation, two pathophysiologic landmarks of diffuse alveolar damage during ARDS, we designed an animal model to understand the mechanisms of the effects of halogenated agents on lung injury and repair. After general anesthesia, tracheal intubation, and the initiation of mechanical ventilation, ARDS was induced in piglets via the intratracheal instillation of hydrochloric acid. Then, the piglets were sedated with inhaled sevoflurane or isoflurane using an ICU-type device, and the animals were ventilated with lung-protective mechanical ventilation during a 4 h period. During the study period, blood and alveolar samples were collected to evaluate arterial oxygenation, the permeability of the alveolar-capillary membrane, alveolar fluid clearance, and lung inflammation. Mechanical ventilation parameters were also collected throughout the experiment. Although this model induced a marked decrease in arterial oxygenation with altered alveolar-capillary permeability, it is reproducible and is characterized by a rapid onset, good stability over time, and no fatal complications.
We have developed a piglet model of acid aspiration that reproduces most of the physiological, biological, and pathological features of clinical ARDS, and it will be helpful to further our understanding of the potential lung-protective effects of halogenated agents delivered through devices used for inhaled ICU sedation.

We describe a model of hydrochloric acid-induced acute respiratory distress syndrome (ARDS) in piglets receiving sedation with halogenated agents, isoflurane and sevoflurane, through a device used for inhaled intensive care sedation. This model can be used to investigate the biological mechanisms of halogenated agents on lung injury and repair.

重症监护病房型装置在猪模型急性呼吸窘迫综合征中的卤化剂递送

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent ...