Special thanks to the Gidday lab for their work in developing the RHP protocol, as well as the Neuro-Models Facility (UTSW) for their assistance in the tMCAo surgeries. This work was supported by grants from the American Heart Association (AMS), The Haggerty Center for Brain Injury and Repair (UTSW; AMS), and The Spastic Paralysis Research Foundation of the Illinois-Eastern Iowa District of Kiwanis International (JMG).

注:此协议被批准的机构动物护理和使用委员会（IACUC）在得克萨斯大学西南医学中心这为健康（NIH）的政策实验动物的使用遵循全国学院。 1.重复缺氧预处理<ol><li>定制设计上的气体调节器4流量计和附加到标准15升感应腔与PVC管，以允许压缩气体从氧气（O 2）的罐，以经由入口端口流入所述腔室。看装备和材料有关自定义设计的更多细节。 </li><li>小鼠分成2组:重复缺氧预处理（RHP）集团，该集团接受暴露于8％和11％O 2，和对照组，其中接收暴露于21％O 2（室内空气）兼任。请参考表1中的频率变化，强度（图8和11％的O 2或21％的O 2），和RHP暴露的持续时间（2或4小时）。&lt;/ LI&gt; <li>删除每个笼子的顶部过滤器盖板，将笼，食物和水的瓶子完好，在连接到各自的O 2坦克室。关闭并固定室的盖。 <ol><li>打开该罐的主气阀，并设置为每个感应腔室的初始流量为每分钟（LPM）的2L曝光的前5分钟。降低流速为1 LPM对于曝光的其余部分。 </li><li>在曝光结束时，减少流至0 LPM和关闭气体阀门的坦克。 </li><li>打开盖子室和更换滤芯顶盖上的每个笼子里。地方标准的住房笼，直到下一次低氧暴露。 </li></ol></li><li>喷绒与NPD（Steris的），或者在每次使用后的等效消毒/除臭每个感应室。 </li><li>在一天两周的过程中的同时露出两个21％和RHP小鼠如表1所述。 </li></ol> 2.瞬态大脑中动脉闭塞（tMCAo） <ol><li>见讨论关于中风的时机下最终RHP曝光更多细节。 </li><li>准备一个无菌的手术工作。清洁周围的工作场所，用70％乙醇或等效消毒和高压灭菌的所有外科手术工具。 <ol><li>设置了激光多普勒血流仪（LDF）仪器测量脑血流量（CBF）的相对变化。打开加热垫至37℃。打开孵化器34℃。 </li></ol></li><li>麻醉小鼠一短暂暴露于4％的异氟醚/ 70％ 的 NO 2/30％O 2的混合在一个小感应腔室。轻轻捏爪子确认适当的麻醉。如果鼠标撤回它的爪子，返回鼠标的感应室，继续异氟醚曝光。 </li><li>从麻醉诱导室取出小鼠和快速插入鼠标的鼻子的鼻锥。打开气流到鼻锥，封闭流向ANESthesia感应室。 <ol><li>在不改变70％ 的 NO 2/30％O 2的混合气体，异氟醚固定在1.8％的维持剂量为程序的剩余部分。呼吸要保持缓慢，在整个过程中定期但是如果呼吸变得急促而浅，增加了异氟醚剂量。维持剂量可以在实验中使用的设备的制造和动物之间变化。 </li></ol></li><li>使用microshaver，剃除毛发上的时间区域中的眼睛的角部和所述耳以及颈部腹侧中线之间。去除多余的皮毛和应用眼部润滑剂用无菌棉签保持角膜的过程中干燥。擦拭用酒精垫和棉签providone碘用无菌棉签切口区保持无菌条件。 </li><li>根据啮齿类动物外科手术指导管理止痛药。 </li><li>通过眼睛和耳朵之间的时间皮肤做一个切口。暴露颞肌。手术采用微型剪刀，剪颞肌韧带在白肌条纹区域内的时间山脊。 <ol><li>轻轻横向推肌肉体积用钳子以可视化的大脑中动脉（MCA）通过头骨。颞肌切开后，该地区可能会充满血液。轻轻用棉签止住任何潜在的出血。 </li><li>目标的LDF探头端部与马华区。记录选为小鼠之间这方面的变化船只。 </li><li>保持就位并平齐的LDF与颅骨直到稳定红细胞通量被读取并记录该值作为基线CBF。在激光多普勒血流仪理想基线CBF是&gt; 600通量，但是这将随制造商。如果基线CBF是&lt;400磁通，流记录是最有可能从附近的静脉，或探针在目标容器的一个不完整的位置。 </li></ol></li><li>后基线CBF被建立，repositi鼠标上使颈部露出。支持它的头，并保持鼠标在稳定麻醉从鼻锥。 </li><li>建立从略低于下颌到锁骨下腹正中切口。 <ol><li>使用镊子，钝解剖所有浅筋膜，以暴露左颈总动脉（CCA）。由结缔组织和迷走神经分离CCA。 </li><li>永久结扎CCA用6.0丝线缝合。将缝线结扎近端为尽可能地留出足够的空间闭塞灯丝位置。 </li><li>循环和松散的领带绕CCA远端第二丝线缝合6.0的遮挡缝合。要小心，不要堵塞动脉的闭塞长丝随后将通过颈螺纹。 </li><li>使用8×2mm的光微serrafine areterial夹子夹住CCA远端松散绑丝线缝合。轻轻提起CCA钳和做一个小切口纵，因为近端缝合结扎作为可能为3 mm vannas。 </li><li>通过切口螺纹12mm的涂硅6.0轨距尼龙阻塞长丝进入动脉管腔，然后前进它几个毫米。松散收紧阻塞丝的尖端周围的二次宽松丝线缝合，以确保血液流动不压灯丝了CCA和消除动脉夹。 </li><li>在CCA到颈内动脉（ICA）和外部颈动脉（ECA）的第一分叉，螺纹闭塞灯丝到第一分叉的右分支进入ICA.Advance闭塞灯丝9至10.5毫米过去第二丝线缝合到左内颈动脉（ICA）。 </li><li>进入ICA后不久，提前阻塞长丝进入左分支在ICA和pterogopalantine动脉（PPA）之间的第二分叉。苯丙醇胺的可视化是不可能这样继续，直到你觉得与遮挡丝满放置一个温和的阻力。起重ICA用钳子可以帮助长丝更容易线程进入第二birfurcation左支。收紧第二丝线。 </li><li>将鼠标使切口在MCA是可见的。随着LDF设备，确认CBF通过LDF读数受阻。一个成功的闭塞是从基线CBF减少&gt; 80％。 </li><li>完全收紧，双结周围的阻塞长丝第二丝线缝合的时候适当的位置来实现的。如果必要的话，稍微推入或拉出细丝遮挡实现CBF标准成功闭塞（从基线CBF 如 &gt; 80％降低）。 </li><li>关闭颈部和头部开口6.0尼龙缝合线。 </li></ol></li><li>地方小鼠在34℃培养箱为闭塞的持续时间。闭塞的推荐长度是60分钟，但是这对于改变脑血管解剖，25岁，应变依赖差异和injur程度Ÿ所需的（轻度，中度，重度）。确保动物脱落麻醉分钟内恢复意识。 </li><li>重新麻醉动物用异氟醚，如步骤2.3中所述，5分钟的预先定义的闭塞期结束前，打开头皮切口，并确认MCA灌注经颅LDF读数依然减少。如果脑血流量的减少不充分（ 例如，&lt;20％基线CBF），在MCA已经闭塞期间再灌注在一些点和鼠标应排除进一步的实验。 </li><li>打开中线颈切口和松散捆绕CCA第三丝线，远端向第二丝线缝合但近端CCA分叉，以确保外颈动脉（ECA）将保持存活灯丝移除之后。 <ol><li>剪切或解开持有闭塞长丝（ 即第二丝线缝合）结 ​​和撤销封闭丝缓慢。一旦取出，迅速关闭周围的CCA第三丝线从ICA尽量减少血液回流。双结这个缝合关闭切口用6.0尼龙缝合线。 </li><li>重新定位鼠标5分钟再灌注后，以量化的CBF的水平。再灌注成功通常被定义为基线脑血流量&gt; 50％的CBF，但与闭塞流，研究者可以建立自己的标准。如果动物呈现低于基线50％的CBF，则很可能在MCA是"永久"闭塞，因而表示另一研究排除标准。 </li><li>关闭两个切口用6.0尼龙缝合线。根据啮齿动物准则提供盐水，麻醉剂（利多卡因）和抗生素。然而，一些小剂量的抗生素（3毫克/公斤的二甲胺四环素的）已经被发现是神经保护以下中风。26 </li></ol></li><li>经过手术后，老鼠的地方在一个无​​尘，无菌笼子里苏醒的恒温培养箱。提供湿润FOOd或营养水合膳食凝胶补充剂和水的培养皿作为动物将具有受限制的流动性中风。恢复过度术后疼痛和死亡过程中密切监测动物。 </li></ol> 3.伊文思蓝（EB）注射<ol><li>再灌注后注射EB 22小时，并在血液中循环应该为以牺牲和TTC染色前2小时。 </li><li>使注射（在盐水中的2％的EB）使用EB溶液并轻轻混合，在室温下进行。过滤通过滤纸或溶液通过附连到一个小注射器以除去未溶解的EB，并储存在室温下0.2微米的过滤器推。 </li><li>制备所需要的注射EB的量（4毫升/公斤体重）画出染料所需量成0.3毫升的胰岛素注射器用29号针头，并确保该溶液是在室温下<ol><li>采用平底阻挡抑制鼠标。握住尾巴，这样的侧脉是UPPermost。侧脉位于尾部的中心线的两侧。抓住尾巴尖，以保持稳定的老鼠注射。 </li><li>针插入静脉约3.5毫米，小心不要刺穿静脉。确认针头是在静脉通过绘制背面上的注射器和寻找血迹。 </li><li>注入所有的染料在1分钟的过程。如果溶液是进入静脉应该有最小的阻力作为压力施加到注射器。在尾部，四肢，鼠标的眼睛立即变色确认EB成功的全身静脉给药。 </li><li>取下尾针，轻轻地用干净的纱布，以止血施加压力。 </li></ol></li><li>开始计时，当鼠标的皮肤变成蓝色。允许在EB循环2小时穿透减弱血脑屏障。 </li></ol> 4. 2,3,5-氯化三苯基四唑（TTC）染色</P&gt; <ol><li>灌注和TTC染色应该出现再灌注后24小时。 </li><li>准备处死前的指定时间为2％TTC解决方案。添加10克TTC粉末的500ml的0.01M磷酸盐缓冲盐水（PBS），pH 7.4中。在水浴热溶液至37℃，以方便的TTC的增溶。 注意:TTC粉和解决方案是一种皮肤，肺，眼睛有刺激性。处理这些材料时，穿戴合适的个人防护装备。 <ol><li>一旦粉末完全溶解后，立即转移至瓶中，盖以箔，并储存在4℃。 TTC和组织沾上TTC是对光敏感。 </li></ol></li><li>在tMCAo后24小时和EB给药后2小时时，牺牲动物提供在一个小感应室中的异氟醚过量。灌注应立即开始下sacricice以尽量减少自溶它开始于以下死亡缺氧的情况下。 </li><li>快速保护动物在泡沫塑料平台前臂通过爪子固定。从正下方的胸腔的中线切穿过腹壁的横向切口。通过隔膜小心切割以暴露心脏。 <ol><li>开始灌流泵以5毫升/分钟的流速用60毫升注射器填充用冰冷的0.01M PBS中并连接到一个27瓜哥翅输液针。将针约0.5cm到心脏的左心室的点尖和剪切右心房。逐步稀释静脉血应该灌注过程中流出的中庭，直到静脉血出现无色。 Transcardially灌注30毫升0.01M磷酸盐缓冲盐水（PBS）通过心脏。 </li></ol></li><li>加入5毫升的TTC溶液成透明20闪烁瓶。 </li><li>紧随灌注，杀头的动物和解剖出用小剪刀和锅铲，如果必要的大脑。检查大脑，排除动物，经历了蛛网膜下腔hemorrhaGE在Willis环，二次缝合位置。检查对侧半球闭塞马华出现面色苍白，没有明显的EB渗漏或水肿。 </li><li>倾的PBS成丙烯酸大脑基质旨在使鼠脑的1.0mm厚的冠状切片。放置脑，背侧，进入基质，并立即倒入PBS在大脑。养脑湿润。 <ol><li>通过插入一个不锈钢0.21毫米厚刀片成从矩阵的喙侧的第二插槽中取出嗅球。 </li><li>通过从基体的尾侧上的第四时隙中插入一个刀片除去小脑。 </li><li>插入一个刀片成在基质中的剩余的槽的中间槽。插入其余刀片，均匀地平分剩余组织，以确保在切片过程中最均匀分布的组织。 </li><li>一旦所有的刀片已插入，添加1〜2滴PBS中至脑滋润它。 </li><li>卸下刀片■一个在从基质与延髓区域开头的时间。保持前7切片TTC分析tMCAo后。用小铲小心从叶片到TTC填充小瓶转移的切片。 </li></ol></li><li>毕竟部分是在小瓶中，盖住它，并在温水浴放置，直到部分变成粉红色。轻轻转动瓶子在浴如有必要，以避免部分重叠，这可能导致不均匀的染色。然后处置TTC和倒4％多聚甲醛溶液放入小瓶覆盖脑切片终止TTC化学反应。 </li><li>立即安排在一个干净的1"×3"搏命的部分和定向部分从吻端至尾。 <ol><li>当该部分被布置在滑动，使用标准扫描器扫描的幻灯片。设置分辨率最低为600 dpi的图像分析。一定要包括动物的名称和扫描图像中的米尺。 </li><li>翻转过来幻灯片和扫描背侧，以确保收集的所有数据。 </li></ol></li></ol> 5.脑梗死体积的定量<ol><li>通过使用标准图像分析软件（ 例如，ImageJ的）量化梗塞体积。 </li><li>扫描图像以高分辨率（ 如 600 DPI）进行充分的分析。裁剪图像。标准化规模为用米尺包括扫描图像中的所有图像。 </li><li>计算使用下面的公式对侧半球的总体积。重复此公式计算同侧半球的总体积。 每个片X层厚总对侧半球的总和</li><li>计算间接梗死体积。 。控制中风同侧水肿使用健康，对侧作为对照27使用下面的公式来计算间接梗死体积: 对侧大脑半球的总体积 - （总volum3次测量梗死电子同侧半球的体积 - 平均） </li></ol> 6.血脑屏障（BBB）完整性定量<ol><li>为了准备EB定量，先掂量2.5英寸称量皿。记录重量并标记2称量皿每个动物:一个用于同侧半球，一个用于对侧半球。 </li><li>扫描TTC染色切片后，平分每个部分用一次性刀片到同侧和对侧半球。将所有7个部分同侧半球一掂船，记录重量。重复对侧半球。 </li><li>紧接重量船转移到烘箱组为56℃48小时。 </li><li>称取干燥的部分。转移两个半球到单独的1.5 ml离心管中。 <ol><li>计算所需要的每一个半球甲酰胺的量（8毫升/克干组织），并添加到它们各自microcentrifugË管。甲酰胺是对光敏感，覆盖铝箔全部甲酰胺处理的样品，从这个点开始。 </li><li>转移离心管至培养箱设定为56℃另外48小时。 </li></ol></li><li>经过48小时，吸取了蓝色上清到另一组标记的离心管中。推组织到管的底部，以最大限度地提取上清液的体积。保持上清液管和处置所有组织。 </li><li>在甲酰胺制备的EB指数系列稀释的标准曲线。包括通过EB的甲酰胺64微克/毫升1空白（甲酰胺只），然后用10指数0.125溶液微克/毫升。 <ol><li>吸液管将300μl标准曲线所作的稀释液到96孔板中。移液器将300μl上清液成相应的孔中。 </li><li>测量在分光光度计上在620nm的吸光度。 </li><li>与吸收Ø比较EB稀释的标准曲线f显示上清样品。的光密度成正比血脑屏障的完整性。 </li><li>假定对侧半球作为背景的​​光密度，并使用式（同侧-对侧）/对侧，以确定倍数变化。有关统计分析，进一步的细节参见Martin 等人 ，2010年18 </li></ol></li></ol>

The authors have nothing to disclose.

单次暴露于全身缺氧（ 即人力资源，2 11％O 2）在小鼠"瞬时"免受tMCAo，29大脑意义缺氧预处理挑战后生反应是持续时间短，基线表型内恢复天。缺氧预处理刺激重复演示大大延长神经保护型的持续时间。6，许多研究表明，频率，幅度和持续时间的反复刺激训练都是这种反应的关键因素。例如，简单地重复同样的强度和缺氧的持续时间（2小时的11％的氧气）每?...

成人残疾和死亡的第四大病因的主要原因，中风是美国面临的成人人口中最衰弱的疾病之一。卒中1动物模型允许对减少缺血性损伤的新方法和实验研究改善中风后恢复。一个新的途径，例如翻译研究的预处理。预处理是有意使用的非破坏性的刺激，以减少从随后，和更严重的，损伤的破坏。2缺氧预处理已经显示出产生在大脑中的多效性的变化提供保护，防止中风在体内和体外研究3，当然 ，单次曝光缺氧只提供短期的神经保护作用，诱导少于72小时耐受缺血的成年小鼠的4连四个星期后的14小时日常暴露在低压缺氧，林等人。 FOUND的神经保护作用只持续了一个星期。5重复缺氧预处理（RHP）的特点是频率，持续时间和低氧暴露强度随机变化。在对比一个单一预处理挑战，RHP诱导脑保护表型，持续长达八周的小鼠。6 RHP减少梗死体积，血-脑屏障（BBB）的破坏，血管炎，和白细胞血细胞渗出数周的最后低氧暴露后。 RHP特别通过降低T细胞，单核细胞，和巨噬细胞群体，同时维持B细胞群体中的缺血性半球炎症减少了缺血性脑7实际上，RHP任何CNS损伤诱导小鼠免疫表型之前，包括中风。 RHP处理的B细胞从RHP处理健康小鼠中分离表现出独特的抗炎表型，具有两个抗原呈递和抗体产生的下调。该在促炎症性免疫机制的整体减少使得RHP极好方法，以诱导内源免疫抑制不仅CNS特异性炎性疾病，而且全身的损伤或疾病的模型，其中包括一个促炎性病理。 RHP降低了梗死体积和血脑屏障破坏下一个暂时性大脑中动脉闭塞（tMCAo）。中风的动物模型，如常用的tMCAo，显着地提高中风的病理生理学的了解，以及更有效的neurotherapeutics设计。首先由小泉等人开发的。，1986年，8 tMCAo过程是诱导中风在啮齿类动物和优选的方法为以下灌注调查炎症一个的广泛使用的方法。作为方法tMCAo演进，更多的近期使用的有机硅涂覆的长丝进一步减少相对于其他模型9,10 &lt;蛛网膜下腔出血的风险/ SUP&gt;，提高了可靠性，但不幸的tMCAo往往产生于梗死体积变化很大。11-13这些研究大多描绘的冠状脑切片梗死区与2,3,5-三苯基氯化（TTC）染色，被认为是金标准梗死定量，因为它是产生生动的，可复制的结果的简单和廉价的方式。 TTC用作存在于线粒体脱氢酶的底物。当脑切片暴露在TTC溶液中，TTC选择性考虑活细胞，其中其非水溶性还原产物，甲，沉淀到深红色存活线粒体。因为在缺血组织线粒体功能障碍的，这个组织保持白色，使受损和健康组织的分化。14 RHP也减少了在缺血半球血脑屏障破坏。6因此，血脑屏障完整性的双重定量同一B内部降雨作为TTC基梗塞体积测定15将提供大约源性保护的充分发挥药效的有用信息，并在未处理的和处理的动物血脑屏障破坏和梗死之间潜在的因果关系。外周血通过具有破坏血脑屏障，继发于中风，潮增加白细胞群，促炎性细胞因子，氧化应激，血管性水肿，和出血性转化中的缺血性半球，最终增加了感染和死亡的发生率在缺血性脑卒中患者16,17测量血脑屏障损伤的动物模型中的常用方法是通过定量的伊文思蓝（EB）的染料泄漏入脑。15,18-21 EB选择性地结合血清白蛋白，球状蛋白质（MW = 65 kDa）的不穿过BBB在未受伤的动物。22继缺血性中风，EB渗入脑和荧光在620nm，允许光密度withi的测量n中的灌注损伤实质22的光密度成正比血脑屏障的时EB已经洗出的验尸皮质脉 ​​管由transcardiac灌注的渗透性。随着动物EB管理TTC染色大脑的即时处理，既梗死体积和血脑屏障破坏，可以有效地量化。应当指出，然而，神经元损伤和血脑屏障破坏不是在中风后大脑伴随工序，23,24这样的牺牲的时间的选择是一个重要的考虑因素。 接下来的协议细节的RHP方法，该tMCAo方法用于诱导临时动脉闭塞该模型中脑动脉闭塞在人类患者，和双组织学方法，用于确定神经和血管中风损伤端点。 TTC测量细胞死亡和累积的组织损伤，从而允许整体梗塞体积的量化乌梅，而电子束提供的血脑屏障损伤的半球定量。

<table><tbody><tr><td>Flowmeters, regulators</td><td>VetEquip, Inc</td><td>Specialty order</td><td>Four flowmeters are attached to 6.0 mm flexible PVC tubing which connects to the inlet port on each induction chamber with a plastic female connector. These flowmeters are bolted to a 6.5&quot; x 1&quot; x 1&quot; metal bar. This metal bar is bolted to a MI-246-P pressure gauge with a DISS outlet. This pressure gauge and flowmeter equipment can be attached to each new gas cylinder with a wrench.</td></tr><tr><td>21% O&lt;sub&gt;2&lt;/sub&gt; tank</td><td>AirGas</td><td>OX USP200</td><td></td></tr><tr><td>11% O&lt;sub&gt;2 &lt;/sub&gt;tank</td><td>AirGas</td><td>Specialty order</td><td></td></tr><tr><td>8% O&lt;sub&gt;2&lt;/sub&gt; tank&amp;nbsp;</td><td>AirGas</td><td>Specialty order</td><td></td></tr><tr><td>15 L induction chambers</td><td>VetEquip</td><td>941454</td><td></td></tr><tr><td>Moor Laber Dopper Flow&amp;nbsp;</td><td>Moor Instruments&amp;nbsp;</td><td>moorVMS-LDF1-HP</td><td>0.8 mm diameter probe&amp;nbsp;</td></tr><tr><td>High Intensity Illuminator&amp;nbsp;</td><td>Nikon</td><td>NI-150</td><td></td></tr><tr><td>Zoom Stereo Microscope&amp;nbsp;</td><td>NIkon</td><td>SMZ800</td><td>Other surgical microscopes may be used.&amp;nbsp;</td></tr><tr><td>Kent Scientific Right Temperature CODA</td><td>Kent Scientific Corporation</td><td>Discontinued</td><td>Recommended replacement is PhysioSuite with RightTemp Temperature Monitoring and Homeothermic Control (Kent Scientific, #PS-RT).</td></tr><tr><td>Hovabator Incubator</td><td>Stromberg&#039;s</td><td>2362-E</td><td>Our model is the 2362N. 2362E is a later model and includes an electronic thermostat.&amp;nbsp;</td></tr><tr><td>V010 Anesthesia system&amp;nbsp;</td><td>VetEquip</td><td>901807</td><td>Includes: ten foot high-pressure oxygen hose, frame, flowmeter, oxygen flush assembly, vaporizer, breathing circuit, chamber, nosecones, waste gas evacuation tubing and two VapoGuard filters</td></tr><tr><td>250 ml isoflurane&amp;nbsp;</td><td>Butler Schein</td><td>NDC-11695</td><td></td></tr><tr><td>D-6 Vet Trim Animal Cordless Trimmer&amp;nbsp;</td><td>Andis&amp;nbsp;</td><td>#23905</td><td>Replacement blades are available from Andis (#23995)</td></tr><tr><td>Betadine&amp;nbsp;</td><td>Fisher Scientific</td><td>19-898-867&amp;nbsp;</td><td></td></tr><tr><td>Q-tips</td><td>Multiple sellers&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td></td></tr><tr><td>Gauze Pads</td><td>Fisher Scientific</td><td>67622</td><td></td></tr><tr><td>Surgical drape</td><td>Fisher Scientific</td><td>GM300&amp;nbsp;</td><td></td></tr><tr><td>Silk Sutures&amp;nbsp;</td><td>Look/Div Surgical Specialties</td><td>SP115</td><td></td></tr><tr><td>Nylon Sutures</td><td>Look/Div Surgical Specialties</td><td>SP185</td><td></td></tr><tr><td>Durmont #5 forceps (2)&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>11251-35</td><td>Angled 45&amp;deg;</td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools&amp;nbsp;</td><td>14028-10</td><td></td></tr><tr><td>3 mm Vannas</td><td>Kent Scientific Corporation</td><td>INS600177</td><td>Straight blade</td></tr><tr><td>Hartman Hemostats&amp;nbsp;</td><td>Fine Scientific Tools</td><td>13002-10</td><td></td></tr><tr><td>Occluding filaments</td><td>Washington University</td><td>Specialty order</td><td>Filaments are silicone coated at Washington Univeristy and provided to UTSW facilities for a fee.&amp;nbsp;</td></tr><tr><td>Evans Blue</td><td>Sigma Aldritch</td><td>E2129-10G</td><td></td></tr><tr><td>Filter Paper&amp;nbsp;</td><td>Sigma Aldritch</td><td>WHA1001150</td><td>150 mm, circles, Grade 1&amp;nbsp;</td></tr><tr><td>Weigh Boats</td><td>Fisher Scientific</td><td>02-202-101</td><td>2.5&quot; diameter</td></tr><tr><td>0.9% Sodium Chloride Injection USP&amp;nbsp;</td><td>Baxter Pharmaceutics&amp;nbsp;</td><td>2B1321</td><td></td></tr><tr><td>0.3 cc insulin syringe with 29 gauge needle</td><td>Becton Dickinson Labware</td><td>309301</td><td></td></tr><tr><td>Flat bottom restrainer&amp;nbsp;</td><td>Braintree Scientific&amp;nbsp;</td><td>FB&amp;nbsp;M</td><td>2.0&quot; diameter</td></tr><tr><td>TTC</td><td>Sigma</td><td>T8877</td><td></td></tr><tr><td>10x PBS, pH 7.4</td><td>Fisher Scientific</td><td>BP399-20</td><td></td></tr><tr><td>Water Bath</td><td>Multiple sellers&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td>Scintillation tubes with TTC may be manually held under running warm water as an alternative to the water bath.</td></tr><tr><td>Styrofoam board</td><td>Multiple sellers&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td></td></tr><tr><td>Large Syringe Kit</td><td>PumpSystems Inc</td><td>P-SYRKIT-LG</td><td></td></tr><tr><td>Perfusion Pump</td><td>PumpSystems Inc</td><td>NE-300&amp;nbsp;</td><td></td></tr><tr><td>60 cc syringe</td><td>Fisher Scientific</td><td>NC9203256</td><td></td></tr><tr><td>27 gauge winged infusion set</td><td>Kawasumi Laboratories, Inc</td><td>D3K1-25G 1</td><td></td></tr><tr><td>20 ml scintillation vial</td><td>Fisher Scientific</td><td>50-367-126</td><td></td></tr><tr><td>Stainless steel spatula</td><td>Fisher Scientific</td><td>14-373-25A</td><td></td></tr><tr><td>Alto acrylic 1.0 mm mouse brain, coronal</td><td>CellPoint Scientific&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td></td></tr><tr><td>0.21 mm stainless steel blades, 25 pk</td><td>CellPoint Scientific&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td>Reusable cryostat blades are an inexpensive alternative.</td></tr><tr><td>4% paraformaldehyde</td><td>Santa Cruz Biotechnology&amp;nbsp;</td><td>SC-281692</td><td></td></tr><tr><td>Superfrost microscope slides&amp;nbsp;</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>HP Scanjet G4050</td><td>Multiple sellers&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td>Other commercial scanners are suitable for this step in the protocol.</td></tr><tr><td>ImageJ&amp;nbsp;</td><td>National Institute of Health</td><td>Catalog number not available&amp;nbsp;</td><td></td></tr><tr><td>Analytical Balance</td><td>Mettler Toledo&amp;nbsp;</td><td>XSE 205U</td><td></td></tr><tr><td>Precision Compact Oven&amp;nbsp;&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>PR305225M</td><td></td></tr><tr><td>1.7 ml microcentrifuge tubes (Eppendorfs)</td><td>Denville Scientific&amp;nbsp;</td><td>C2170</td><td></td></tr><tr><td>Formamide</td><td>Fisher Scientific</td><td>BP228-100</td><td></td></tr><tr><td>96-well plates</td><td>Fisher Scientific</td><td>07-200-9</td><td></td></tr><tr><td>Epoch Microplate Spectrophotometer&amp;nbsp;</td><td>BioTek&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td></td></tr></tbody></table>

quantification of neurovascular protection following repetitive hypoxic preconditioning and transient middle cerebral artery occlusion in mice

中风的实验动物模型是了解中风的病理和开发更有效的治疗策略非常宝贵的工具。 2周协议重复缺氧预处理（RHP）诱导长期保护，防止中枢神经系统（CNS）损伤的局灶性缺血性脑卒中的小鼠模型。 RHP由9随机暴露于缺氧该变化在两个持续时间（2或4小时）和强度（8％和11％的O 2）。 RHP减少梗死体积，血 - 脑屏障（BBB）的破坏，并为周后的最后暴露于缺氧的中风后的炎症反应，这表明了长期的感应的内源性的CNS保护表型。对于梗死体积和血脑屏障破坏的双重定量的方法是有效的评估小鼠RHP或其他神经保护剂推测神经血管的保护。成年雄性Swiss Webster小鼠被RHP或持续时间相当于曝光预处理到21％氧气<suB&gt; 2（ 即室内空气）。 60分钟短暂的大脑中动脉闭塞（tMCAo）诱导后2周的最后低氧暴露。无论是缺血再灌注经颅激光多普勒血流仪证实。二十二个小时再灌注后，伊文思蓝（EB）进行静脉内，通过尾静脉注射给药。 2小时后，将动物用异氟烷过量和脑切片处死沾满2,3,5-氯化三苯基四唑（TTC）。梗死体积再量化。接着，EB从组织中提取超过48小时tMCAo后确定血脑屏障破坏。总之，RHP是一个简单的协议，它可以被复制，以最少的成本，以诱导中风损伤小鼠长期内源性神经血管保护，与平移潜力其它CNS基和全身促炎疾病状态。

这项研究包括25男瑞士韦伯斯特小鼠为10周龄时随机开始进入RHP（N = 10）或21％O 2（N = 15）组。两周后的最终RHP曝光，外科手术进行，以盲基和天之间平衡。继tMCAo，1个鼠标术后恢复过程中死亡和1个鼠标被排除在研究，因为它不符合再灌注CBF标准。排除两种小鼠在21％O 2组。按照ARRIVE准则，28表2示出的外科参数。间接的梗死体积，半球肿胀（ 即 ，水肿），和伊文思?...

Watch this Scientific Journal Video about Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice at JoVE.com

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

神经血管保护定量继在小鼠重复缺氧预处理和瞬态大脑中动脉闭塞

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

quantification-neurovascular-protection-following-repetitive-hypoxic

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

13.7K 次观看.  University of Texas Southwestern Medical Center. This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

视频: Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

双侧颈总动脉闭塞足够的预处理刺激诱导局灶性脑缺血早期缺血耐受

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

科研

医学

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2血管闭塞/低血压:全脑缺血模型大鼠

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

横向慢性颅窗口准备启用<em&gt;在体内</em&gt;观察继末节小鼠大脑中动脉闭塞

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

神经科学

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

对大脑中动脉闭塞再灌注损伤诱发中风小鼠的手术方法

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

缺血性中风和缺血再灌注小鼠的诱导使用梗塞区的中心动脉闭塞技术和可视化

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO2 during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO2, or by mice inhaling 20% CO2. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.

实验模型研究酸性后处理对脑缺血的神经保护作用

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

Assessing Neuroprotective Effects of Glycyrrhizae Radix et Rhizoma Extract Using a Transient Middle Cerebral Artery Occlusion Mouse Model

Ischemia followed by reperfusion of cerebral blood flow after a stroke leads to the death of nerve cells and loss of brain tissue. The most commonly used animal model for studying stroke is the middle cerebral artery occlusion (MCAO) model. Previous research studies have reported different infarct sizes even when the same experimental animal species was used under similar MCAO conditions. Therefore, we developed an improved experimental method to address this discrepancy. Mice were subjected to MCAO using a filament as the occlusion material to mimic human stroke conditions and filament thickness was optimized to establish more reproducible infarction volume. Mice treated with a methanol extract of Glycyrrhizae Radix et Rhizome (GRex) following stroke induction showed a significantly decreased total infarction volume and increased number of surviving cells relative to the untreated control group. This modified experimental protocol successfully and reproducibly demonstrated the beneficial effect of GRex on ischemic stroke.

In this study, we modify an existing experimental method to obtain more reproducible results, by establishing a middle cerebral artery occlusion (MCAO) mouse model. Oral administration of Glycyrrhizae Radix et Rhizome (GR) methanol extract (GRex), following stroke induction, significantly decreased total infarction volume relative to the untreated control group.

应用短暂的中脑动脉闭塞小鼠模型评价甘草提取物的神经保护作用

Ischemia followed by reperfusion of cerebral blood flow after a stroke leads to the death of nerve cells and loss of brain tissue. The most commonly used ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

脑缺血再灌注的双血管闭塞小鼠模型

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

由瞬态双边常见胡萝卜动脉闭塞引起的缺血性视网膜损伤的 Murine 模型

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

大脑中动脉闭塞的短暂性脑缺血模型

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

作者聚焦：通过大脑中动脉闭塞模型评估缺血性卒中损伤 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

脑卒中短暂性中动脉闭塞模型

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...