The authors would like to acknowledge support through the American Heart Association (10SDG2630139), the Welch Foundation and through the NIH Director&#8217;s New Innovator Grant (1DP2 OD008716-01). The authors would like to thank the services provided by the ICMB (Institute of Cellular and Molecular Biology) core facility and TherapeUTex at University of Texas at Austin.

注:在本协议中显示的所有方法已被批准的机构动物护理和使用委员会。 1.制备手术台的<ol><li>成立加热垫与循环上手术台温水，解剖显微镜下。放置在不锈钢底板上的加热垫。放置在基板和加热垫无菌吸水垫。 </li><li>安排在相邻的无菌吸水垫无菌手术工具。收集两对成角度的细尖镊子，两对成角度的镊子，三止血钳，2拉钩电线，小手术剪，微型剪刀，1血管成形术线（0.15英寸直径）的6.0丝缝合3 6英寸的段。 </li><li>弯曲成形术线的圆端以匹配股动脉的曲率。这将使它更容易在手术过程中推进线。 </li></ol> 2.制备鼠标手术<ol><li>连续吸入2.5％异氟醚麻醉的小鼠。确保整个手术过程监控动物的状态。执行鼠标的脚的压紧测试，以确认其完全麻醉。确保动物时夹送测试施用不移动。 </li><li>涂抹润滑软膏动物的眼睛，以防止干燥。固定动物在仰卧位使用外科胶带的吸收垫。继续通过鼻锥管理2.5％，异氟醚。 </li><li>使用脱毛霜，去除腿部和腹部的皮毛到中线。用清水彻底冲洗皮肤。脱毛可以事先手术来进行一天。需要注意的是过度治疗与奶油可能导致皮肤刺激。 </li><li>紧接外科手术前，应用碘伏到脱毛区域用棉签消毒皮肤。冲洗脱毛皮肤用70％乙醇和干用无菌棉签。重复3次。 </li><li>通过皮下注射施用手术前剂量为5毫克/公斤卡洛芬的。 </li></ol> 3.隔离股动脉<ol><li>使用小手术剪，使一个弧形切口，在皮肤上的股动脉。用拉钩牵开磁性固定器，找到股动脉解剖直言和保护周围组织。滋润定期用生理盐水组织灌溉用消毒棉签应用生理盐水。 </li><li>隔离使用镊子的股动脉。轻轻地用细尖镊子维管束分隔神经。避免穿刺静脉，不损伤神经。按神经离束，以避免刺激它。 </li><li>轻轻分开从股动脉股静脉，定位股骨分叉。分叉的区域是特别困难剖析。 </li><li> Posterior分叉，环股动脉和安全用止血钳下一个6.0丝缝线。这个近端缝线将用于限制在动脉的血流。 注:在执行结扎方法与烧灼方法时有一个在关系略有变化（ 见图1和图2）。 </li><li>远端分叉，股动脉下循环6.0丝线缝合，安全用止血钳。此远端缝合辅助中动脉的定位。 </li><li>环2在股动脉的肌支缝合线，预配合他们，并用止血钳固定。记住滋润组织用生理盐水。如果执行丝损伤的烧灼方法中，只有一个环缝合线是必要的肌肉分支。 </li></ol>股动脉线损伤4.性能<ol><li>通过拉动近端缝合限制血流进入股动脉。稍微拉远hemost在和止血钳固定支暴露部位的动脉切开术。通过把缝线周围结扎肌支。 注:向上提升的动脉会更有效地限制水平比单独拉血流量。 </li><li>切断小分支两缝线之间的烧灼。采用微型剪刀，在分叉的侧枝执行动脉切开术。使用镊子外科带的辊平衡以稳定微剪刀。 </li><li>确认用细尖镊子动脉切开术的存在。轻轻抬起与钳动脉切开开幕。电线的圆形端引入使用镊子的动脉切开术。为缓解丝的前进，一或两滴利多卡因添加到使用注射器的区域。 </li><li>当线到达近侧缝合，松开缝合线，并调整它，以便它不能妨碍导线的进步。插入金属丝，直到它不能进一步前进。次的尖端Ë线应停止在腹股沟韧带的区域。 </li><li>允许金属丝保持在股动脉一分钟。一分钟后，缩回和推进所述导线在锯切运动十次伤害和剥夺股动脉的内皮。对于更低的严重程度的损伤，减少的次线被拉动和流出动脉的数目或离开丝在动脉仅1分钟。 </li><li>拉回金属丝缓慢。当线的圆端已通过近端缝合，通过拉动近端缝合限制流进动脉。完全缩回线。 </li></ol> 5.结扎肌支<ol><li>拧紧肌支剩余的缝合。这将防止从动脉切开出血。回流到肌支，并确认血液不能从中泄漏。修剪从缝合线两端的肌支。 </li></ol> 6.替代方法:本地化驹动脉切开术的terization 注:另一种方法，可采取避免肌肉分支结扎，并允许通过较大主股动脉血管通路。 <ol><li>从4.1开始，通过拉动近端缝合限制流动到股动脉。拉远侧缝合和缝线固定肌肉分支略微露出的部位的动脉切开术（ 图1和图2）。 </li><li>采用微型剪刀，在股动脉和肌肉分支的分支点执行动脉切开术。如果切口是在动脉的侧面，它可以更容易地烧灼。使用镊子外科带的辊平衡以稳定微剪刀。 </li><li>为了测试烧灼的成功，通过松开近端缝线恢复流动到股动脉。如果发生在动脉切开出血，反复烧灼。如果烧灼是成功的，血流将被重新存储远端的动脉切开术。 </li><li>引入线和在第4节进行导线损伤。 </li><li>继续限制血流进入股动脉。热火细尖烧灼至少6英寸远离鼠标。由于烧灼尖端冷却，轻轻地应用它侧动脉切开术，以关闭切口。 </li><li>为了测试烧灼的成功，通过松开近端缝线恢复流动到股动脉。如果发生在动脉切开出血，反复烧灼。如果烧灼是成功的，血流将恢复远端的动脉切开术。除去临时近端和远端的关系。 </li></ol> 7.植入血管周围给药的补丁<ol><li>创建血管周围药物贴剂可如先前的研究11或通过使用类似的方法描述来创建。生硬的药物递送贴片的使用无菌剪刀阻挡区域的角</li><li>放置药物递送轻拍CH到受伤的股动脉与药物释放面朝动脉。必要时，可使用产钳来提高贴片的位置。 </li></ol> 8.伤口愈合和恢复<ol><li>关闭伤口用一个简单间断缝合组成的平方海里。不连续缝合延长伤口愈合的情况下动物尝试删除缝合。 </li><li>关掉麻醉，并从该组向上取出动物。让动物恢复的变暖垫。 </li><li>继续关注鼠标的复苏。检查切口部位每一天，以确保它保持闭合。用于治疗手术后疼痛，管理一个皮下注射5毫克/公斤的卡布洛芬，每12小时的手术后，然后每12小时2天。如果疼痛持续超过了最初的2天，请教兽医进一步止痛药的方向。 </li></ol> 9.收获股动脉组织学<ol><li> 28天手术后，对小鼠进行二氧化碳安乐死。 注:二氧化碳的适当流速应置换为每分钟腔的10-30％，并会根据所使用的腔室的大小而异。切割膜片和心脏穿刺的应执行安乐死的次级方法。 </li><li>确保鼠标在使用医用胶带仰卧位。做一个切口在大腿动脉，其中的初始手术切口制成。 </li><li>对于这两个受伤的动脉和对侧的腿未受伤的动脉，直言解剖和安全的使用环境拉钩和磁牵引器固定器，找到股动脉组织。滋润定期用盐水灌溉的组织。使用无菌棉签申请盐水。小心不要损坏动脉。 </li><li>后股动脉已被隔离从原来的动脉切开的部位的腹主动脉，扎赛格换货附近的动脉切开的原址丝线缝合的。此缝合将有助于确定股动脉的最远端和缓和处理样品。 </li><li>使用显微解剖剪切除股动脉。使一个切口远侧的缝合。使上股动脉的相对端的另一切口，腹主动脉旁。 </li><li>切除的动脉转移到含有生理盐水的玻璃培养皿。进一步剖析动脉，去除多余的结缔组织和脂肪。轻轻地用生理盐水冲洗，从管腔清除血液。 </li><li>动脉转移到10％的福尔马林缓冲液的小瓶。储存小瓶在4℃下轻轻摇动48小时。 </li><li>固定动脉转移到70％的乙醇，以存储直到它被处理用于组织学。 </li><li>嵌入在石蜡块和部分染色块的动脉。 </li><li>进行组织化学和免疫组织化学染色评估损伤内膜HYP程度erplasia。 注:对于代表的结果，我们采用HE染色，以可视化的核和整体形态或Movat的Pentachrome染色可视化的弹性薄片和其他动脉成分。 </li></ol>

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We have presented a method for performing vascular injury in mice and delivery therapeutic compounds to the injured region through a perivascular cuff. The ligation method for femoral and carotid arteries has been described in conventional methods papers and characterized extensively11-14,19-23 and we present an alternative method for achieving the same vascular injury that is less technically demanding procedure that can often be used in younger mice. One of the chief advantages of the using a mouse wire injury model with a perivascular patch is that it allows the use of genetically modified mice or murine models of disease. The endovascular manipulation of small arteries is technically demanding and this aspect has motivated the creation of models that create injury through a number of extravascular methods including ligation,11,24,25 perivascular cuffing26 and electric shock.27 In our procedures, a contralateral sham surgery can be used as a control by performing the protocol without the wire injury or, alternatively, non-operated control arteries can be used. The critical steps within the protocol are the following: (1) the surgical cut down and dissection of the femoral artery from surrounding structures, (2) the formation of the arteriotomy, (3) performance of the wire injury, (4) closure of the arteriotomy and (5) implantation of the perivascular cuff. Below we will review the major steps and address potential pitfalls in their performance. 
For the surgical cut down, the major challenge is the separation of the femoral artery from the femoral vein. Care should be taken at this stage, as it is easy to cause bleeding during the separation and the vein tears easily in comparison to the artery. Using forceps to blunt dissect and remove adventitia surround the artery and vein can help this process (see techniques shown in the video). Also, the artery may have branches underneath that can be torn if an overly aggressive technique is used. 
For the formation of the arteriotomy and wire injury, it is critical to use high quality microsurgical scissors. It is often helpful to cut at a 45-degree angle to the axis of the artery and to stabilize the cut with forceps as shown in the video. Too much or too little tension on the artery can make cutting difficult and it is sometimes helpful pull the distal tie before the proximal tie to fill the artery with blood to aid in the performing the arteriotomy. After the cut, opening the hole with the closed point of the scissors or forceps can aid in allowing entry of the wire. The size of the arteriotomy should cut approximately half way through the artery. Again, too much tension on the artery will make insertion of the wire difficult and may tear the vessel. A low to moderate level of tension should be used with the ties. When inserting the wire a slight twisting motion can be helpful but too much twisting will cause the artery to seize, preventing advancement of the wire.
A key step in performing our alternative method is the controlled local cautery that seals the vessel without blocking flow. The cautery should be heated away from the tissue and then touched to the side of the artery. Heating with the cautery while touching the vessel will burn surrounding tissues including the vein and femoral artery branches. One potential limitation of this technique is that if the arteries are not consistently cauterized there may be variations in the flow following the surgery or in the amount of inflammation caused by the burn. Analysis of the artery away from this region and consistent technique can minimize the potential impact of these limitations. We hope this alternative method will make the wire injury surgery more accessible and reduce the need for older mice for vascular injury studies.

Arterial injury and inflammation caused by angioplasty and stent implantation can induce neointimal hyperplasia that contributes to the thickening of the arterial wall, a process known as restenosis.1,2 The formation of restenosis is major mode of failure for interventions such as angioplasty and stenting with bare metal stents.3 Due to recent concerns with the inhibition of vascular healing in arteries treated with drug eluting stents, there is also a need to find compounds that can inhibit restenosis while maintaining vascular healing and re-endothelialization.4-7 In addition, while stents have had success in the coronary vasculature, percutaneous interventions of all types in the peripheral arteries continue to fail at a higher rate due to restenosis.8-10 Mouse models of surgical interventions allow the use of powerful genetic manipulations that can provide mechanistic insight into the mechanisms underlying the failure of clinical therapies and can provide an initial test bed for compounds to inhibit intimal hyperplasia. 
Here, we describe a mouse model of vascular injury that allows the testing of therapeutic compounds to inhibit neointimal hyperplasia and assess whether their effects on re-endothelialization following endothelial denudation. A key challenge in executing vascular injury in mice is the technical skill needed to obtain vascular access and to restore flow to the injured artery following the wire injury. For this reason, simple arterial ligation models have been used to study neointimal hyperplasia in mice that do not require endovascular manipulations but are easier to implement.11 However, this type of surgical model differs substantially from the mechanical and biological aspects of a percutaneous intervention, lacking key aspects including arterial wall stretch, endothelial denudation and luminal blood flow following injury. 
We present two methods for obtaining and closing vascular access for wire injury of the femoral artery in mice. The first technique is the conventional method described by several groups previously and uses vascular access through a side branch of the femoral artery.12-14 This method requires older, larger mice and more surgical skill to implement the endoluminal access through the smaller artery. It also requires the ligation of the muscular branch of the femoral artery following the procedure. The second method we describe uses an arteriotomy in the branch point of the main and side branch and thereby allows for a larger access to the artery for performing wire injury. In this method, the arteriotomy is closed using controlled local cauterization that leaves both branches with blood flow following the procedure. The conventional method is applicable to mice of at least 20 weeks of age while the alternative method can be used in mice of at least 15 weeks of age. In both methods, the wire creates arterial stretch and abrasion leading to injury and endothelial denudation. Following either procedure a perivascular drug delivery patch can be implanted that allows the delivery of compounds to alter the response to injury. The use of the drug delivery patch allows mice to be used as a test bed for new compounds to inhibit restenosis through perivascular therapies.15,16

<table><tbody><tr><td>Straight spring wire, 0.15&amp;rdquo; diameter</td><td>Cook</td><td>G02426</td><td></td></tr><tr><td>High Temperature cautery</td><td>Bovie Medical Corp.</td><td>HIT1</td><td></td></tr><tr><td>High-temperature fine tip for cautery</td><td>Bovie Medical Corp.</td><td>H101</td><td></td></tr><tr><td>Micro-scissors</td><td>Fine Science Tools</td><td>15000-13</td><td>For performance of arteriotomy</td></tr><tr><td>Angled fine-tipped forceps</td><td>Fine Science Tools</td><td>11251-35</td><td>For blunt dissection of vascular bundle</td></tr><tr><td>Angled forceps</td><td>Roboz</td><td>RS-5069</td><td>For clearing tissues</td></tr><tr><td>Surgical scissors</td><td>Roboz</td><td>RS-5840</td><td>For cutting skin</td></tr><tr><td>Retractor</td><td>Fine Science Tools</td><td>18200-10</td><td></td></tr><tr><td>Retractor wire</td><td>Fine Science Tools</td><td>18200-05</td><td>Attached to retractor</td></tr><tr><td>Base plate</td><td>Fine Science Tools</td><td>18200-03</td><td>For use with retractor</td></tr><tr><td>Magnetic retractor fixator</td><td>Fine Science Tools</td><td>18200-01</td><td></td></tr><tr><td>Needle Holder</td><td>Roboz</td><td>RS-7822</td><td></td></tr><tr><td>Hemostatic forceps</td><td>Biomedical Research Instruments, Inc.</td><td>34-1000</td><td></td></tr><tr><td>Dissecting microscope</td><td>Meiji Techno</td><td>EMZ-5TR</td><td></td></tr><tr><td>Microscope light source</td><td>Meiji Techno</td><td>FT191</td><td></td></tr><tr><td>Warm water recirculator</td><td>Gaymar</td><td>TP-500</td><td>For maintaining mouse body temperature</td></tr><tr><td>Reusable heating pad</td><td>Gaymar</td><td>TP-R 22G</td><td>For maintaining mouse body temperature</td></tr><tr><td>Lidocaine</td><td>Various</td><td></td><td></td></tr><tr><td>4.0 Vicryl suture with half circle needle</td><td>Ethicon</td><td>J494G</td><td>For post-surgical wound closure</td></tr><tr><td>Sterile cotton-tipped applicators</td><td>Puritan</td><td>25-806-2WC</td><td>For application of depilatory cream and absorbing fluids</td></tr><tr><td>Depilatory cream</td><td>Nair</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Various</td><td></td><td></td></tr><tr><td>Betadine</td><td>Various</td><td></td><td></td></tr><tr><td>70% ethanol</td><td>Various</td><td></td><td></td></tr><tr><td>6.0 braided silk suture</td><td>Teleflex Medical</td><td>4-S</td><td>For isolation of femoral artery during surgery</td></tr><tr><td>0.9% sodium chloride</td><td>Various</td><td></td><td>For irrigating tissues</td></tr><tr><td>Gel eye lubricant</td><td>Various</td><td></td><td></td></tr><tr><td>Glass Petri dish</td><td>Pyrex</td><td>3160-60</td><td>For femoral artery harvest</td></tr><tr><td>10% buffered formalin</td><td>Various</td><td></td><td>For fixation of femoral artery</td></tr><tr><td>70% ethanol</td><td>Various</td><td></td><td>For fixation of femoral artery</td></tr><tr><td>Bouin&#039;s fluid</td><td>Electron Microscopy Sciences</td><td></td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alcian blue, 1%</td><td>Electron Microscopy Sciences</td><td>26385-01</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alkaline alcohol</td><td>Electron Microscopy Sciences</td><td>26385-02</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Orcein, 0.2%</td><td>Electron Microscopy Sciences</td><td>26385-03</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Hematoxylin alcoholic, 5%</td><td>Electron Microscopy Sciences</td><td>26385-04</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Ferric chloride, 10%</td><td>Electron Microscopy Sciences</td><td>26385-05</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Lugol&#039;s Iodine</td><td>Electron Microscopy Sciences</td><td>26385-06</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Woodstain scarlet-acid fuchsin working solution</td><td>Electron Microscopy Sciences</td><td>26385-07</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Acetic acid, 0.5%</td><td>Electron Microscopy Sciences</td><td>Various</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Phosphotungstic acid, 5%</td><td>Electron Microscopy Sciences</td><td>26385-09</td><td>For Movat&#039;s Pentachrome staining</td></tr><tr><td>Alcoholic saffron, 6%</td><td>Electron Microscopy Sciences</td><td>26385-10</td><td>For Movat&#039;s Pentachrome staining</td></tr></tbody></table>

murine model of femoral artery wire injury with implantation of a perivascular drug delivery patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

以下电线损伤，内膜增生的发展随着时间的推移和后14〜28天，通常检验。在这项工作中铅的小鼠描述健壮代内膜增生， 如图组织学结果的技术的3。一种未受伤的股动脉将展示完整的弹性薄片和一个正常的厚度和周长。受伤的股动脉将显示内膜增生，降低弹性片层和展示再内皮化，在以后的时间点。再内皮通常完全在约21天，但此取决于所使用的小鼠的背景应变，并且可以使用免疫染色对切片内皮标记物，或优选，测得的上连接使用伊文思蓝染料脸制剂进行评估，扫描电子显微镜免疫组化或与连接的动脉面对准备共聚焦显微镜，17,18对于内膜增生量化它最好是测量内膜面积或内膜到媒体比在三个地点沿受伤动脉占沿动脉的损伤区域的长度的电位变化。 <img alt="图1" src="/files/ftp_upload/52403/52403fig1highres.jpg" /> 图1:这两种方法用于线损伤的股动脉的关键步骤的照片的步骤，为结扎法（顶部）向烧灼方法（底），用于执行丝损伤 ​​的比较。在连接法中，两个缝线固定肌肉分支是预先打结，以便它们可以被收紧结扎肌肉分支。两个肌支缝线的更远端被紧固。动脉切开术和电线插入在肌肉分支执行。之后进行的损伤，对肌肉分支剩余缝线收紧和修整。在烧灼的方法，只有一个Suture是根据肌支循环。动脉切开术和电线插入是在肌肉分支从股动脉分支点执行。在执行完损伤，切口无烧灼或者结扎股动脉或肌支。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52403/52403fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52403/52403fig2highres.jpg" /> 图2:这两种技术的电线受伤的关键步骤高倍照片为结扎法（顶部）向烧灼方法（底）的步骤特写比较。动脉切开术的位置划分的虚线。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52403/52403fig2large.jpg" target="_blank">请点击此处查看本一个更大的版本</a>数字。 <img alt="图3" src="/files/ftp_upload/52403/52403fig3highres.jpg" /> 图3:电线受伤从动脉部分中的过程的组织学在14天后代表性的组织学结果用苏木精和曙红（上）和Movat的pentachrome染色（底部）。标记图像中有内腔（L），内膜（I）和弹性层（EL）。

Watch this Scientific Journal Video about Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch at JoVE.com

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

股动脉线损伤的小鼠模型的血管周围给药补丁植入

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

murine-model-femoral-artery-wire-injury-with-implantation

Nanyang Technological University

Research

JoVE Journal

Medicine

17.9K 次观看.  University of Texas at Austin. We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis. 

视频: Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

编程干细胞的治疗性血管新生使用可生物降解高分子纳米微球

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

科研

生物工程

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

颈动脉支架植入再狭窄的研究的小鼠模型

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

医学

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

无线电遥测系统，以监测大鼠脊髓横断和胚胎神经干细胞移植在心血管功能

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

动脉再狭窄小鼠模型:股导线损伤的技术方面

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

水凝胶构造和纤维蛋白基胶方法来提供治疗的小鼠心肌梗死模型。

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导:"电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, we developed a rat model of patch angioplasty that can be used in either a vein or an artery, creating a patch venoplasty or a patch arterioplasty, respectively. Technical aspects of this model are discussed. The infra-renal IVC or aorta are dissected and then clamped proximally and distally. A 3 mm venotomy or arteriotomy is performed in the infrarenal inferior vena cava or aorta of 6 to 8 week-old Wistar rats. A bovine pericardial patch (3 mm x 1.5 mm x 0.6 mm) is then used to close the site using a 10-0 nylon suture. Compared to arterial patches, venous patches show increased neointimal thickness on postoperative day 7. This novel model of pericardial patch angioplasty can be used to examine neointimal hyperplasia on vascular biomaterials, as well as to compare the differences between the arterial and venous environments.

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

血管补片在大鼠主动脉和下腔静脉

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

心肌梗塞穆林模型中的 3D 生物打印贴片移植

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...