这项调查得到了美国国立卫生研究院 （NIH） 国家心肺血液研究所 （NHLBI） 的支持，编号为 1R01HL149404-01A1 （BL），以及威斯康星大学麦迪逊分校心血管研究中心 （JB） 的 Ruth L. Kirschstein 国家研究服务奖 T32 HL 007936。使用 Biorender.com 创建或编辑地物。使用 GraphPad Prism 10 软件进行统计分析。

动物规程由威斯康星大学麦迪逊分校机构动物护理和使用委员会 （M005792） 批准。
1. 动物维护
<ol><li>在标准维护食物上饲养老鼠。使用成年小鼠或年轻成年小鼠 （8-12 周龄）。 注意:使用成体可确保动物完全成熟，并限制主动脉直径变化可能与动物生长有关的任何机会。在这项研究中，我们使用了手术时年龄为 22-24 周的雄性和雌性 C57BL/6J 小鼠。Lu 及其同事未观察到年轻和老年小鼠之间的动脉瘤反应存在显著差异8。此外，虽然大多数动脉瘤建模是在雄性小鼠中进行的，但该模型成功地在雄性和雌性小鼠中诱导了 AAAs9。</li>
	<li>确定研究持续时间并将动物分配到治疗组或假（对照）组。给治疗组小鼠施用 0.2% BAPN 饮用水，并让它们进行手术，局部施用活性弹性蛋白酶到肾下主动脉。给对照动物施用未经处理的水，并将它们进行手术，将变性弹性蛋白酶应用于肾下主动脉。</li>
</ol>2. 开始使用补充 B-氨基丙腈 （BAPN） 的饮用水
<ol><li>手术前两天，在 0.2% BAPN 饮用水中开始治疗小鼠。制备大量 BAPN 水，并在 4 °C 的黑暗中储存长达 28 天。在给老鼠喂食之前，请确保 BAPN 水达到室温。 注意:我们建议在整个研究期间每 7 天更换一次笼子中的 BAPN 水。</li>
</ol>3. 手术当天准备材料
<ol><li>将手术手套剪成 5 mm x 10 mm 的条状，稍后用于帮助在用弹性蛋白酶处理之前隔离主动脉。通过在手术单的中心切一个 ~ 1.5 cm x 3 cm 大小的椭圆形来准备手术单。展开 2 英寸 x 2 英寸的纱布并切成两半，制成大约 2.5 厘米 x 10 厘米的纱布条，以便稍后用于缩回腹内容物。高压灭菌所有手术器械（参见 材料表）并设置无菌手术区域，如图 1 中的示例所示。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66812/66812fig01.jpg"> 图 1:为 AAA 的弹性蛋白酶/BAPN 小鼠模型做准备的无菌手术设置示例。 缩写:BAPN = ß-氨基丙腈;AAA = 腹主动脉瘤。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66812/66812fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<ol start="2"><li>将干净的笼子放在加热灯下，并将盐水放在灯附近以加热至体温 （37 °C），以准备术后恢复笼。确保加热灯安全放置，使回收笼和盐水温暖但不超过 37 °C。 打开水泵，开始通过加热垫循环温水。</li>
</ol>4. 动物手术准备
<ol><li>将小鼠置于诱导室中，并使用低流量电子蒸发器以 200 mL/min 的速度用 5% 异氟醚麻醉它们。麻醉后，称量每只小鼠并皮下注射 0.6 mg/kg 丁丙诺啡缓释剂和 20 mg/kg 卡洛芬用于镇痛。使用电动毛发修剪器夹住腹部的毛皮，从下腹部到剑突的下部。使用纱布或实验室湿巾刷掉多余的毛发。将小鼠放回笼子中，等待至少 20 分钟让镇痛生效，然后再进行手术。</li>
	<li>镇痛后至少 20 分钟后，将小鼠置于麻醉诱导室中，并使用低流量电子蒸发器再次以 200 mL/min 的速度输送 5% 异氟醚，直至小鼠被镇静。</li>
	<li>从诱导室中取出镇静小鼠，并将其仰卧在手术区域上。涂抹眼部凝胶并用手术胶带固定鼻锥。以 50 mL/min 的速度将递送的吸入异氟醚降至 1-2% 的维持率。用手术胶带固定鼠标的前爪和后爪。</li>
	<li>检查小鼠的小腹是否有膀胱。用拇指、食指和中指轻轻地对膀胱施加外部压力以诱导排尿;同时，用一块纱布吸走尿液。 注意: 小心不要污染手术区域。</li>
	<li>首先用含碘或洗必泰的磨砂膏和棉签用 70% 的酒精对腹部进行消毒。从腹部中央开始，以圆周运动 3 倍向外进行。在两次应用之间让该区域短暂干燥。</li>
	<li>检查是否缺乏脚趾捏合反应，以确保麻醉充分。确保鼻锥和四肢固定。将手术单放在鼠标上，开口直接位于手术准备的腹部上。 注意:请勿在鼠标上拖动窗帘，以避免潜在的污染。</li>
</ol>5. 手术诱导 AAA
<ol><li>进入腹腔:<ol><li>洗手并戴上干净的丁腈或无菌外科手套。在接触手术区域之前，请务必在手套上喷洒 70% 乙醇，并将戴手套的手揉搓至干燥。</li>
		<li>使用钝镊子在中线腹部的皮肤上搭帐篷。用手术剪刀在皮肤上划一个小口，然后纵向延长切口，大约 2-3 厘米长。</li>
		<li>使用镊子抬起直肌，以识别半透明的白线。用剪刀通过白线进入腹腔，然后沿着白线向近端和远端延伸。</li>
	</ol></li>
	<li>暴露腹主动脉:<ol><li>用温盐水蘸湿一条纱布和两根棉签。将纱布的一端紧紧卷成一半，留下一条宽大的尾巴，制作腹部卷。</li>
		<li>使用皮肤牵开器回缩右腹壁。</li>
		<li>使用湿润的棉签，轻轻地将小肠和大肠扫到左上腹，进行右内侧内脏旋转，并观察主动脉和下腔静脉 （IVC）。使用腹部卷将肠道缩回视野之外 – 将纱布卷起的一端塞入肠道下方，然后将尾端绕出身体，轻轻地包裹肠道。轻轻拉紧纱布的尾部，使肠道远离视野。调整腹卷和皮肤牵开器以获得腹膜后器官的最佳视图，如图 2A 所示。 注意:腹卷有助于保持肠道湿润并防止其被手术器械意外损坏。确保纱布在手术过程中保持湿润，以防止肠道干燥。小心不要用力缩回肠道，因为这会导致肠系膜上动脉和肠脉管系统扭结，从而可能导致缺血性损伤。此外，最初清扫小肠时，要小心大肠和下肝（肝结肠韧带）之间的薄半透明附件，如果不小心，很容易撕下肝包膜并导致出血。如果在回缩过程中这条韧带有张力，请用剪刀将其急剧分开。</li>
	</ol></li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66812/66812fig02.jpg"> 图 2:腹部回缩的表示和小鼠肾下主动脉暴露的最佳手术视图。 （A） 放置纱布腹卷有助于回缩腹腔内器官，而相对的牵开器有助于提供腹膜后可视化。将无菌手术单 （透明以显示动物方向） 放在麻醉的动物上，以帮助保持无菌。（B） 腹膜后筋膜（绿色框）前部覆盖主动脉。（C） 腹膜后筋膜夹层后肾下主动脉的示例。主动脉与 IVC 的分离可以通过从主动脉和 IVC 之间的潜在空间开始，该空间位于左肾静脉的远端，因为它向前穿过（黄色圆圈）。缩写:IVC = 下腔静脉。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66812/66812fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<ol start="3"><li>肾下主动脉的周向夹层和分离术:<ol><li>确认 IVC 和肾下主动脉在全视野中。首先进入并分开腹膜后 （RP） 筋膜，开始暴露主动脉（图 2B）。确定沿肾下前主动脉平行的性腺（睾丸或卵巢）动脉（图 2B 和 图3）。使用镊子钝地分开性腺动脉之间的筋膜，并继续纵向向前暴露主动脉（图 2C）。 注意:RP 筋膜是一层薄而半透明的结缔组织，包含淋巴管和内脏神经丛。有必要通过 RP 筋膜解剖以暴露主动脉。但是，不要通过主动脉外膜的结缔组织进行解剖。外膜（白色结缔组织）的撕裂会暴露出介质（呈亮红色），一旦应用弹性蛋白酶，主动脉可能会在该部位破裂。</li>
		<li>接下来，开始将腹主动脉从 IVC 中分离出来。从 IVC 和主动脉之间的一个小间隙开始解剖，该间隙位于左肾静脉穿过主动脉时下缘的正下方（图 2C）。使用镊子的尖端轻轻地分开主动脉和 IVC 之间的结缔组织纤维，并继续在此水平围绕主动脉周向工作。 注意:IVC 壁非常薄，通过一层细小的纤维结缔组织紧密粘附在主动脉上。小心避免处理 IVC 或尽可能清洁 IVC。首先从 IVC 上解剖主动脉的右侧（然后再从周围的肌肉组织中解剖主动脉的左侧）将有助于主动脉从 IVC 上"脱落"。</li>
		<li>继续直截了当地解剖主动脉和 IVC 之间的平面，向主动脉分叉处尾部工作。一旦到达主动脉分叉处，就停止进一步的远端夹层。 注意:在解剖肠系膜下动脉 （IMA） 周围时要格外小心，IMA 通常位于肾下主动脉中段附近，并横向穿过 IVC。</li>
		<li>一旦主动脉的右缘与 IVC 分离，向近端返回到左肾静脉的水平。从主动脉左外侧缘解剖 RP 筋膜，沿周周工作，直到主动脉完全隔离。有关腹膜后夹层的相关解剖结构，请参见 图 3 。 注意:仔细解剖主动脉后面，因为腰静脉和动脉的位置和数量变化很大。有关这种解剖出血风险高的区域的参考，请参见 图 4 。</li>
		<li>仔细检查主动脉是否尽可能与 IVC 和周围肌肉组织圆周隔离，仔细解剖由 IMA 和腰动脉拴系的主动脉段周围。</li>
		<li>沿着主动脉的左右边缘放置一条手套，如图 5A 所示。尝试尽可能多地覆盖 IVC。</li>
		<li>使用手持式卡尺测量最宽的主动脉直径并记录 3 次测量结果。在测量前后用 70% EtOH 喷洒卡尺的尖端。避免用卡尺尖端直接接触主动脉，以防止污染。 注意:也可以使用使用经过校准的相机显微镜拍摄的照片。</li>
	</ol></li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66812/66812fig03.jpg"> 图 3:小鼠下腹部、骨盆和腹膜后血液供应的解剖结构。 缩写: R = right;L = 左。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66812/66812fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66812/66812fig04v2.jpg"> 图 4:腹膜后夹层和肾下主动脉周向隔离期间损伤和出血高风险部位。 缩写: L = 左;IMA = 肠系膜下动脉。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66812/66812fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66812/66812fig05.jpg"> 图 5:弹性蛋白酶/BAPN 小鼠 AAA 模型期间对弹性蛋白酶应用或假手术的术中反应。 （A） 在应用弹性蛋白酶之前，将手套段沿主动脉的长度放置，以帮助保护 IVC 和肠道免受弹性蛋白酶的影响，同时保持主动脉浸泡在弹性蛋白酶中 （B） 应用变性弹性蛋白酶不会导致主动脉扩张（蓝色框）。最大主动脉直径在基线时测得 0.627 mm，然后在局部变性弹性蛋白酶 5 min 后测得 0.607 mm。（C） 治疗 5 分钟后应用弹性蛋白酶导致主动脉扩张。在这个例子中，主动脉（绿色）从 0.607 毫米扩张到 0.953 毫米，直径增加了 57%。缩写:BAPN = ß-氨基丙腈;AAA = 腹主动脉瘤。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66812/66812fig05large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<ol start="4"><li>弹性蛋白酶应用:<ol><li>使用棉签轻拍主动脉上多余的血液或液体。</li>
		<li>接下来，将一块 10 毫米 x 2 毫米的干纱布放在主动脉顶部。使用移液管分配 5 μL 弹性蛋白酶（或对照变性弹性蛋白酶）以使纱布和主动脉饱和。轻轻地将手套的碎片折叠在主动脉周围。 注:要制备用于假组或对照组的变性弹性蛋白酶，请将弹性蛋白酶在 100 °C 下煮沸 30 分钟。</li>
		<li>让弹性蛋白酶作用于主动脉 5 分钟。在此潜伏期内，如果需要，释放腹卷和皮肤牵开器施加的一些张力。 注意:由于弹性蛋白酶的批量效应，我们鼓励研究人员在给定研究中的所有实验中使用同一瓶弹性蛋白酶。对于每一瓶新的弹性蛋白酶，我们建议进行剂量反应，以确保没有大量的早期破裂（4 周之前）。弹性蛋白酶应用的持续时间也可以在 4 到 6 分钟之间调整，具体取决于对弹性蛋白酶的反应。</li>
		<li>5 分钟后，重置肠道回缩并展开手套的碎片。用 1 mL 温热的 0.9% 无菌生理盐水冲洗腹腔，同时小心地去除主动脉上的纱布和手套碎片。用 10 厘米 x 10 厘米的纱布吸收腹部的盐水。重复冲洗腹部，总共 3 x 3 mL。</li>
		<li>使用手持式卡尺重新测量弹性蛋白酶应用后最宽的主动脉直径并记录 3 倍。参见图 5B，C 有关用假和活性弹性蛋白酶治疗的主动脉扩张的例子。 注意:弹性蛋白酶前后的三次测量值的平均值可用于计算治疗后主动脉直径的百分比变化。通常，弹性蛋白酶处理后立即出现明显的扩张 ~30-50%，这有助于确保弹性蛋白酶功能正常且主动脉得到充分治疗。主动脉的直径不应随着变性弹性蛋白酶的应用而改变，或者尺寸可能略小（可能是由于痉挛）。</li>
	</ol></li>
	<li>腹腔闭合:<ol><li>小心地从肠道下方取出腹卷并排出体外。如果需要，在肠道中涂抹额外的盐水，以防止在取出时粘在腹卷上。检查以确保肠道呈粉红色并充分灌注。 注意:没有必要尝试将肠道重新定位回其原始位置;尝试这样做可能会有肠扭动或内疝的风险。</li>
		<li>使用正在运行的 5-0 不可吸收单丝缝合线重新接近腹部筋膜。用 3-4 个皮肤钉缝合皮肤。</li>
	</ol></li>
</ol>6. 术后动物护理
<ol><li>将鼠标放入带有加热灯的恢复笼中。确保笼子的温度是温暖的，而不是热的。</li>
	<li>给予 0.5-1 mL 0.9% 生理盐水的皮下液体推注。</li>
	<li>让鼠标在温暖的笼子中自行恢复 ~20 分钟，直到按照机构方案激活，然后返回笼子。</li>
	<li>根据机构方案，术后第 1 天手术后 24 小时施用卡洛芬 20 mg/kg，并每天持续 3 天。</li>
</ol>7. 主动脉测量和组织收获
<ol><li>使用异氟醚和宫颈脱位实施安乐死后，重新打开腹腔。通过胸骨延伸切口以进入胸部。切除右心房，在 2 分钟内用 10 mL 冷的 1% DPBS 溶液灌注左心室。切除肺、肝和脾。 注意:小心不要伤到肠道;肠道内容物溢出会影响组织分析。</li>
	<li>如上所述，暴露腹主动脉，并测量最大肾下主动脉直径。继续解剖整个主动脉和心脏。一旦心脏和主动脉被分离，切开所有动脉分支和髂总动脉，在主动脉上留下完整的短段。将心和主动脉放在标尺和图像旁边的对比色背景上。</li>
</ol>8. 数据分析和报告
<ol><li>为了帮助解决人为错误，使用手持式卡尺时，每次测量主动脉直径至少 3 次，然后将直径报告为平均值。</li>
	<li>将 AAA 定义为健康主动脉直径增加 50%。确保在研究结果中包括总主动脉直径和直径百分比变化。</li>
</ol>

评估弹性蛋白酶治疗后小鼠主动脉瘤的发育

<ol>
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W., Wang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+murine+models+of+ruptured+abdominal+aortic+aneurysm.">Progress in murine models of ruptured abdominal aortic aneurysm.</a> Front Cardiovasc Med. 9, 950018 (2022).</li><li>Lu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chronic+advanced+stage+abdominal+aortic+aneurysm+murine+model.">A novel chronic advanced stage abdominal aortic aneurysm murine model.</a> J Vasc Surg. 66 (1), 232-242 (2017).</li><li>Berman, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+aortic+aneurysm+severity+and+growth+depend+on+topical+elastase+concentration+and+lysyl+oxidase+inhibition.">Experimental aortic aneurysm severity and growth depend on topical elastase concentration and lysyl oxidase inhibition.</a> Sci Rep. 12 (1), 99 (2022).</li><li>Deaths, percent of total deaths, and death rates for the 15 leading causes of death in 5-year age groups, by race, and sex. Centers for Disease Control and Prevention Available from: <a target="_blank" href="https://www.cdc.gov/nchs/nvss/mortality/lcwk1.htm">https://www.cdc.gov/nchs/nvss/mortality/lcwk1.htm</a> (2015)</li><li>Dansey, K. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+abdominal+aortic+aneurysms.">Mouse models of abdominal aortic aneurysms.</a> Arterioscler Thromb Vasc Biol. 24 (3), 429-434 (2004).</li><li>Busch, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+mouse+models+of+abdominal+aortic+aneurysm+to+the+translational+needs+of+vascular+surgery.">Translating mouse models of abdominal aortic aneurysm to the translational needs of vascular surgery.</a> JVS Vasc Sci. 2, 219-234 (2021).</li><li>Anidjar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastase-induced+experimental+aneurysms+in+rats.">Elastase-induced experimental aneurysms in rats.</a> Circulation. 82 (3), 973-981 (1990).</li><li>Bhamidipati, C. 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本手稿的作者无需声明任何利益冲突。

了解 AAA 的复杂病理生理学对于改善主动脉瘤疾病的管理至关重要。虽然积极开发新的策略来改善手术结果，但 AAA 在我们的老龄化社会中仍然普遍存在，动脉瘤破裂仍然是美国死亡的主要原因10。因此，AAA 检测、预防和治疗策略方面未满足的需求需要进一步的基础动脉瘤研究11。
准确有效地概括人类 AAA 特征和行为的动物模型对于动脉瘤病理生理学的机制研究和确定潜在的治疗靶点至关重要。虽然目前的动物模型可以模拟人类疾病中发生的动脉瘤变化的主要方面，但没有一个模型可以完全代表人类 AAA 的真实复杂性。目前，小鼠是动物 AAA 建模中最广泛接受的物种。研究人员应考虑每种小鼠模型在其特定动脉瘤研究中的各种优势和劣势，例如 Daugherty 等人和 Busch 等人的评论中专业描述的那些12,13。
Anidjar 等人于 1990 年首次描述了使用弹性蛋白酶在啮齿动物中诱导 AAA14。使用注射泵用猪胰弹性蛋白酶灌注主动脉会产生大约 50% 到 70% 的初始扩张，并且扩张的节段有利地表现出与人类 AAA 相似的病理特征，例如中层变性和外膜炎症。然而，经典的灌注模型可以说是技术上最具挑战性的动脉瘤模型，通常在第二周形成的动脉瘤此后开始逐渐消退。Bhamidipati 等人在 2012 年证明，弹性蛋白酶的外膜应用也可以成功诱导类似的动脉瘤，这些动脉瘤在15 号动脉瘤中更具可重复性。一种挑战性小得多的模型，局部弹性蛋白酶模型在动脉瘤研究中被广泛采用。Xue 及其同事的方法论文中讨论了局部弹性蛋白酶模型的其他方法和优势16。
小鼠 AAA 的弹性蛋白酶/BAPN 模型由 Lu 及其同事于 2017 年8 开发。引入 0.2% BAPN 饮用水改善了对经典局部弹性蛋白酶模型的许多批评，现在产生的动脉瘤不断扩大到 AAA 破裂的程度。在他们 2017 年的研究中，他们证明与弹性蛋白酶组相比，弹性蛋白酶/BAPN 处理组的小鼠具有显着更高的 AAA 形成率（93% 对 65%，P < 0.01），后者也是更晚期的 AAA。在 100 天的研究期间，弹性蛋白酶/BAPN 组中的 AAAs 继续扩张至 >800% 基线直径并形成腔内血栓 （53.8%），46.2% 在实验结束前自发破裂。该模型使研究人员能够研究可能随着时间的推移影响动脉瘤进展和稳定性的因素。
Berman 等人通过改变局部弹性蛋白酶的浓度、研究持续时间、BAPN 给药的时间以及动物性别的影响，进一步探索了弹性蛋白酶/BAPN 模型9。用 5 μL 较高浓度的弹性蛋白酶 （5 mg/mL 或 10 mg/mL） 处理，在 56 天内产生的动脉瘤大于 2.5 mg/mL。腔内血栓形成的患病率也取决于弹性蛋白酶浓度，在 5 mg/mL 治疗的小鼠中为 28.6%，在 10 mg/mL 治疗的小鼠中为 62.5%。他们还证明弹性蛋白酶/BAPN 模型可以在雌性小鼠中诱导动脉瘤。虽然只研究了几只雌性小鼠 （n=5），但他们发现雌性动脉瘤更容易破裂 （5 只小鼠中有 2 只），并且在 56 天时明显大于雄性 AAA。
在本文中，我们旨在提供一种方法来解决手术建模的最大局限性之一，即手术过程的变化。如果对夹层程度和用弹性蛋白酶处理的主动脉面积没有明确的共识，该模型的结果可能会因动物、研究人员和机构而异。我们观察到小鼠之间的许多解剖学变化，包括腰动脉和静脉的数量和大小，以及 IMA 的位置、左性腺静脉的起飞等，这在尝试仅治疗肾下主动脉的一部分或特定段时可能会受到限制。在这里，我们证明，从左肾动脉近端到远端分叉处的肾下主动脉的整个长度的圆周解剖有助于提供可重复的主动脉暴露程度，尽管解剖学存在差异，同时提高动脉瘤诱导的成功率并为操作者提供明确的边界。此外，IVC 的大小和更靠前的位置往往覆盖主动脉的大部分，如果不从 IVC 中分离出来，这会影响治疗的主动脉量。虽然有必要切除腹膜后筋膜以暴露主动脉，但重要的是不要完全解剖主动脉外膜的结缔组织并暴露任何介质层，因为这通常会导致在 5 分钟的弹性蛋白酶潜伏期内破裂。这可以作为对该模型解剖程度的额外内部控制，但在采用该模型时，这可能是一个令人沮丧的学习曲线。操作员还将了解高风险区域（图 4），这些区域在手术过程中很容易受伤并导致无法控制的出血。
虽然该模型的程序步骤保持一致很重要，但研究的持续时间和间期超声检查的时间可能会因研究目标而异。主动脉扩张立即开始使用弹性蛋白酶，但使用该模型的研究通常在手术后 28 天跟踪小鼠7，如本示例实验所示。在研究晚期 AAAs、长期生长、腔内血栓形成或破裂时，应考虑延长研究持续时间。
其他围手术期措施，例如保持动物体温和水合状态，有助于提高这种侵入性手术的动物存活率。在手术期间使用加热垫并放置在温暖的恢复笼中有助于避免体温过低。盐水在用于冲洗腹腔之前应加热。手术后直接皮下推注液体可以解释手术过程中不显性液体流失，并帮助动物在即刻恢复阶段保持足够的水分。通过仔细的组织处理和一致的方法，弹性蛋白酶/BAPN 模型可以由经验丰富的操作员在每只小鼠 30 分钟到 45 分钟之间进行，并可靠地产生 AAA，围手术期并发症非常低。
我们的结果表明，BAPN 与弹性蛋白酶应用前肾下主动脉的圆周解剖相结合会产生大的、不断扩大的 AAA，直径更大，破裂率更短。在该实验中，所有用活性弹性蛋白酶处理的雄性 （6 / 6） 和雌性 （6/6） 小鼠均成功诱导了 AAAs。弹性蛋白酶暴露 5 分钟导致主动脉直径立即增加约 30-40%，这有助于确认治疗组之间弹性蛋白酶应用成功且一致。与 Berman 等人类似，我们已经证明该模型可以在雌性小鼠中诱导 AAAs，雌性小鼠也比雄性小鼠具有更大的破裂反应。一半的雌性小鼠（6 只中的 3 只）在 28 天内破裂，而雄性 6 只中有 0 只破裂，但是，雌性小鼠的体重比雄性轻。雄性小鼠的 AAA 直径增加了 257%，而雄性对照的 -4%，而存活的雌性小鼠的直径增加了 417%，而雌性对照的直径增加了 -16%。由于雌性组破裂次数较多，存活的雄性和雌性治疗小鼠在 28 天时主动脉直径无显著差异。我们推测假小鼠在研究结束时表现出较小的主动脉直径，因为主动脉在初始解剖过程中往往会略微扩张，然后在 28 天时形成疤痕组织。
弹性蛋白酶/BAPN 模型具有一定的局限性。主动脉的周向清扫术需要精细的手术技巧，但有助于提高可复制性和动脉瘤诱导的程度。与局部弹性蛋白酶模型类似，弹性蛋白酶活性也存在批量效应，因此如前所述，在给定实验中为所有动物使用同一瓶弹性蛋白酶非常重要。虽然 AAA 腔内血栓和破裂的发生率随着时间和动脉瘤严重程度的增加而增加，但这些在该模型中无法保证或完全预测。
总之，弹性蛋白酶/BAPN 模型在雄性和雌性小鼠中产生大的、真正的肾下 AAA，这些 AAA 随着时间的推移逐渐扩大，形成腔内血栓，并且能够破裂。这种小鼠模型的这些优势有助于更好地概括人类动脉瘤的一些行为和特征。虽然技术上困难，但仔细彻底地解剖主动脉可以增强动脉瘤反应。目前，弹性蛋白酶/BAPN 方法是研究肾下腹主动脉瘤的高级模型。

动脉瘤被定义为血管的病理扩张大于健康血管直径的 50%1。尽管腹主动脉瘤 （AAA） 是老年人群中常见的疾病，发病率约为 >5% 的男性> 65 岁，但没有治疗 AAA1 的定向治疗策略。目前 AAA 的管理仅限于减少危险因素和根据主动脉直径或生长速度进行开放手术或血管内手术的手术修复2。AAA 的最大危险是动脉瘤破裂，如果不治疗，这是致命的，在这种紧急情况下进行修复可能会带来高达 90% 的死亡风险1。
AAA 的病理生理学复杂、多因素且尚不完全清楚3.人类 AAA 的特征包括主动脉壁的真动脉瘤扩张伴炎症细胞浸润、腔内血栓的存在以及导致最终破裂的进行性扩张 3,4。此外，AAA 与高龄相关，男女占 9:1 的优势，最常见于肾下主动脉5。对动物体内人类 AAA 的所有特征和行为进行建模仍然是一个持续的挑战6。
目前的 AAA 建模主要在小鼠中进行，动脉瘤通常使用以下三种方法之一诱导 - 通过皮下植入的渗透泵输注血管紧张素 II （AngII），以及通过将氯化钙 （CaCl2） 或弹性蛋白酶直接应用于主动脉7。在后一种方法中，猪胰弹性蛋白酶 （PPE） 应用于肾下主动脉的一段，并导致被膜介质弹性薄片内的弹性蛋白纤维酶促降解。这种结构损伤导致主动脉壁变弱和动脉瘤向外扩张。然而，单独使用局部弹性蛋白酶会产生相对较小的肾下动脉瘤，这些动脉瘤不会随着时间的推移而逐渐扩大或破裂。最近，Lu 等人通过对β-氨基丙腈 （BAPN） 进行了改进，这是一种赖氨酰氧化酶的不可逆抑制剂，适用于他们的弹性蛋白酶处理的小鼠8。通过阻止弹性蛋白和胶原纤维的交联，BAPN 补充剂会导致弹性蛋白酶受损的主动脉逐渐扩张至破裂点。弹性蛋白酶/BAPN 模型的 AAA 发病率也高于局部弹性蛋白酶模型，并且产生的动脉瘤也更大，包含腔内血栓8。
在弹性蛋白酶/BAPN 模型中，手术夹层的程度和主动脉对弹性蛋白酶的暴露会影响该模型的成功和可复制性。在本手稿中，我们描述了在环周隔离整个肾下主动脉段后，BAPN 饮用水和局部弹性蛋白酶应用于主动脉的共同给药提高了可复制性，解释了动物之间的解剖学差异，并导致更高的 AAA 诱导率、动脉瘤大小和破裂发生率。在本文中，我们将描述一种使用局部补充弹性蛋白酶和 BAPN 的水的组合可靠地诱导小鼠晚期腹主动脉瘤的标准化方法。

<table><tbody><tr><td>0.5 L induction chamber</td><td>Kent Scientific Corporation</td><td>SOMNO-0530XXS</td><td>anesthesia induction chamber</td></tr><tr><td>0.9% sodicum chloride injection, USP, 20 mL</td><td>Hospira</td><td>NDC 0409-4888-03</td><td>normal saline</td></tr><tr><td>3 mL syringe Luer-Lok Tip with BD PrecisionGlide Needle 22 G x 3/4</td><td>BD</td><td>REF 309569</td><td>syringe, 22 G needle</td></tr><tr><td>3-Aminopropionitrile Fumarate</td><td>TCI</td><td>A0796</td><td>BAPN</td></tr><tr><td>3-Aminopropionitrile Fumarate salt</td><td>Sigma-Aldrich</td><td>A3134-25G</td><td>BAPN</td></tr><tr><td>Avant Delux gauze sponges, 2&quot; x 2&quot; 4-Ply</td><td>Medline</td><td>NON26224</td><td>gauze sponges</td></tr><tr><td>Balding clipper</td><td>Whal Clipper Corporation</td><td>8110</td><td>hair clippers</td></tr><tr><td>betadine surgical scrub (povidone-iodine, 7.5%)</td><td>Avrio</td><td>NCD 67618-154-16</td><td>betadine surgical scrub</td></tr><tr><td>blunt forceps</td><td>ROBOZ</td><td>RS-5130</td><td>blunt forceps</td></tr><tr><td>Buprenorphine ER-lab</td><td>ZooPharm</td><td>BERLAB0.5</td><td>buprenorphine</td></tr><tr><td>carprofen</td><td>Norbrook</td><td>NDC 55529-131-11</td><td>carprofen</td></tr><tr><td>CASTROVIEJO 5.75&quot; straight with lock</td><td>ROBOZ</td><td>RS-6412</td><td>Castroviejo needle driver</td></tr><tr><td>cotton tipped wood applicators, 6&quot;</td><td>Dynarex</td><td>No. 4302</td><td>cotton tipped wood applicators</td></tr><tr><td>DESMARRES 5.5&#039; rectractor&amp;nbsp;</td><td>ROBOZ</td><td>RS-6672</td><td>skin rectractor&amp;nbsp;</td></tr><tr><td>digital caliper, 0-150 mm</td><td>World Precision Instruments</td><td>501601</td><td>digital caliper</td></tr><tr><td>DPBS (1x)</td><td>Gibco</td><td>14190-144</td><td>DPBS</td></tr><tr><td>Elastase from porcine pancrease Type I</td><td>Sigma-Aldrich</td><td>E1250-10MG</td><td>elastase &gt;4.0 units/mg protein</td></tr><tr><td>Ethanol 200 proof</td><td>Decon Labs, Inc</td><td>2701</td><td>ethanol diluted to 70%</td></tr><tr><td>eye lube</td><td>Optixcare</td><td>14716</td><td>eye lube</td></tr><tr><td>Germinator 500 dry sterilizer</td><td>CellPoint Scientific, Inc</td><td>5-1450</td><td>dry bead sterilizer</td></tr><tr><td>heat therapy mat</td><td>Adroit Medical Systems</td><td>V016</td><td>heat therapy mat</td></tr><tr><td>heat therapy pump</td><td>Adroit Medical Systems</td><td>HTP-1500</td><td>heat therapy pump</td></tr><tr><td>isoflurane, USP</td><td>Akorn Animal Health</td><td>NCD 59399-106-01</td><td>isoflurane</td></tr><tr><td>L-10 pipette</td><td>Rainin</td><td>LTS 0.5-10 uL</td><td>pipette</td></tr><tr><td>Low profile anesthesia mask, small</td><td>Kent Scientific Corporation</td><td>SOMNO-0801</td><td>anesthesia nose cone</td></tr><tr><td>micro dissector scissors</td><td>ROBOZ</td><td>RS-5619</td><td>micro dissector scissors</td></tr><tr><td>microscope</td><td>Leica</td><td>S9i</td><td>microscope</td></tr><tr><td>Nii-LED high intensity LED illuminatorLED exertnal light</td><td>Nikon Instruments, Inc</td><td>83359 NII-LED</td><td>external dissection light</td></tr><tr><td>nylon 5-0 monofilament, black non-absorbable suture</td><td>Oasis</td><td>MV-661-V</td><td>5-0 nylon suture</td></tr><tr><td>polyisoprene surgical gloves, GAMMEX Non-Latex PI Micro, size 7.5</td><td>Ansell</td><td>20685975</td><td>non-latex surgical gloves</td></tr><tr><td>Reflex 7 mm stainless steel wound clips</td><td>CellPoint Scientific, Inc</td><td>203-1000</td><td>wound clips</td></tr><tr><td>scale</td><td>Ohaus</td><td>Compass CR2200</td><td>scale</td></tr><tr><td>SomnofFlo Accessory Kit</td><td>Kent Scientific Corporation</td><td>10-8000-71</td><td>tubing for electronic vaporizer&amp;nbsp;</td></tr><tr><td>SomnoFlo electronic vaporizer</td><td>Kent Scientific Corporation</td><td>SF2992</td><td>low-flow electronic vaporizer</td></tr><tr><td>SomnoPath Flow Diverter</td><td>Kent Scientific Corporation</td><td>SP1016</td><td>flow diverter for electronic vaporizer</td></tr><tr><td>SS/45 sharp forceps</td><td>ROBOZ</td><td>RS-4941</td><td>sharp forceps</td></tr><tr><td>surgical scissors</td><td>ROBOZ</td><td>RS-6010SC</td><td>surgical scissors</td></tr><tr><td>vessel forceps</td><td>Dumont</td><td>VES 0.35</td><td>vessel forceps</td></tr></tbody></table>

advanced abdominal aortic aneurysm modeling in mice by combination of topical elastase and oral &#223;-aminopropionitrile

当与补充 β-氨基丙腈 （BAPN） 的饮用水结合时，腹主动脉瘤 （AAA） 的局部弹性蛋白酶鼠模型得到增强，以可靠地产生具有模仿人类 AAA 行为的真正肾下动脉瘤。将弹性蛋白酶局部应用于肾下主动脉外膜会导致主动脉壁弹性层的结构损伤并引发动脉瘤扩张。共同施用 BAPN 是一种赖氨酰氧化酶抑制剂，通过减少胶原蛋白和弹性蛋白交联来促进持续的壁变性。这种组合导致大 AAA 逐渐扩大，形成腔内血栓，并能够破裂。改进手术技术，例如圆周隔离整个肾下主动脉段，可以帮助标准化程序，以实现猪胰弹性蛋白酶的一致和彻底应用，尽管小鼠之间的操作者和解剖学存在差异。因此，弹性蛋白酶/BAPN 模型是一种在小鼠中手术诱导 AAA 的改进方法，它可以更好地概括人类动脉瘤，并为研究动脉瘤生长和破裂风险提供更多机会。

本研究使用 22-24 周龄的雄性和雌性 C57BL/6J 小鼠。肾下主动脉用 5 μL 弹性蛋白酶 （6.9 mg 蛋白质/mL，6 单位/mg 蛋白质） 或变性弹性蛋白酶处理 5 min。与未治疗的基线主动脉直径相比，弹性蛋白酶处理的雄性小鼠在暴露于弹性蛋白酶 5 分钟后主动脉直径增加 43.4%，而处理的雌性主动脉增加 33.6% （P = 0.0342）。假手术的主动脉直径在暴露于变性弹性蛋白酶 5 分钟内表现出相对无变化 （雄性 0.5%;雌性 -2.8%）。12 只接受治疗的小鼠和 6 只假小鼠中没有手术相关的死亡。 表 1 显示了为期 28 天的研究数据。在接受治疗的雌性小鼠中，6 只中有 3 只死于 AAA 破裂;1 例在术后第 20 天，2 例在 25 天（图 6）。接受治疗的男性没有 AAA 破裂。在所有治疗小鼠中成功诱导 AAA （定义为基线主动脉直径增加 >50% 或 AAA 破裂导致死亡） （12 只中的 12 只）。在 28 天时，治疗雄性的平均 AAA 直径为 2.86 ± 0.31 毫米，平均变化百分比变化为 257 ± 54%，而存活的治疗雌性小鼠的 AAA 直径为 3.60 ± 1.87 毫米，平均百分比变化为 417 ± 286%（图 7）。假小鼠的主动脉直径相对没有变化。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66812/66812fig06.jpg"> 图 6:雄性和雌性 B6 小鼠在 AAA 的 28 天弹性蛋白酶/BAPN 模型中的存活率。 （A） 6 只治疗过的雌性小鼠中有 3 只发生 AAA 破裂（20 天时 1 只小鼠，然后 25 天时 2 只小鼠），而 6 只治疗雄性小鼠在 28 天时没有破裂。（B） 死于 AAA 破裂的雌性小鼠尸检的代表性图像。AAA 破裂表现为大的腹膜后血肿（左）和肾下 AAA 伴室壁缺损（右）。缩写:BAPN = ß-氨基丙腈;AAA = 腹主动脉瘤。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66812/66812fig06large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66812/66812fig07.jpg"> 图 7:弹性蛋白酶/BAPN 和假雄性和雌性 B6 小鼠在 28 天时的最大主动脉直径。 （A） 与假小鼠相比，治疗小鼠在 28 天时表现出明显更大的肾下直径。（B） 弹性蛋白酶和 BAPN 的组合在雄性和雌性 B6 小鼠中成功产生大型肾下 AAA。缩写:BAPN = ß-氨基丙腈;AAA = 腹主动脉瘤。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/66812/66812fig07large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="3"></td>
			<td>86 男 假</td>
			<td>86 女性假动作</td>
			<td>86 雄性弹性蛋白酶/8APN</td>
			<td>86 雌性弹性蛋白酶/8APN</td>
		</tr><tr><td colspan="3">小鼠数量</td>
			<td>3</td>
			<td>3</td>
			<td>6</td>
			<td>6</td>
		</tr><tr><td colspan="3">年龄 （周）</td>
			<td>22.3 ± 0.0</td>
			<td>22.7 ± 0.7</td>
			<td>23.1 ± 0.2</td>
			<td>23.2 ± 0.2</td>
		</tr><tr><td colspan="3">体重（g;手术时）</td>
			<td>36.3 ± 2.5</td>
			<td>23.7 ± 1.2</td>
			<td>32.8 ± 1.7*</td>
			<td>23.7 ± 0.8</td>
		</tr><tr><td colspan="3">治疗前主动脉直径 （mm）</td>
			<td>0.89 ± 0.02</td>
			<td>0.75 ± 0.04</td>
			<td>0.81 ± 0.07</td>
			<td>0.73 ± 0.09</td>
		</tr><tr><td colspan="3">治疗后主动脉直径 （mm）</td>
			<td>0.90 ± 0.03</td>
			<td>0.73 ± 0.01</td>
			<td>1.15 ± 0.03**</td>
			<td>0.98 ± 0.12**</td>
		</tr><tr><td colspan="3">治疗 5 分钟后的百分比变化 （%）</td>
			<td>0.5 ± 4.4</td>
			<td>-2.8 ± 5.3</td>
			<td>43.4 ± 10.2***</td>
			<td>33.6 ± 4.5***</td>
		</tr><tr><td colspan="3">AAA 发生率 （%）</td>
			<td>0 / 3</td>
			<td>0 / 3</td>
			<td>6 / 6</td>
			<td>6 / 6</td>
		</tr><tr><td colspan="3">AAA 破裂 28 天</td>
			<td>0 / 3</td>
			<td>0 / 3</td>
			<td>0 / 6</td>
			<td>3 / 6</td>
		</tr><tr><td colspan="3">存活至 28 天</td>
			<td>3 / 3</td>
			<td>3 / 3</td>
			<td>6 / 6</td>
			<td>3 / 6</td>
		</tr><tr><td colspan="3">28 天时的最大主动脉直径 （mm）</td>
			<td>0.85 ± 0.01</td>
			<td>0.64 ± 0.01</td>
			<td>2.86 ± 0.31*</td>
			<td>3.60 ± 1.87**</td>
		</tr><tr><td colspan="3">28 天时主动脉直径变化百分比 （%）</td>
			<td>-4 ± 2</td>
			<td>-16 ± 2</td>
			<td>257 ± 54*</td>
			<td>417 ± 286**</td>
		</tr></tbody></table>表 1:AAA 弹性蛋白酶/BAPN 小鼠模型的 28 天模型结果。 ±*P<0.05、**P<0.005、***P<0.0001 与同性假手术相比，通过单因素方差分析 Fischer 检验。缩写:BAPN = ß-氨基丙腈;AAA = 腹主动脉瘤。

作者聚焦:使用 Elastase 模拟人动脉瘤的增强型小鼠 AAA 模型

该方案描述了一种有方法的手术方法，通过直接将弹性蛋白酶应用于肾下主动脉并通过饮水施用 β-氨基丙腈来模拟小鼠晚期腹主动脉瘤。

局部弹性蛋白酶和口服 β-氨基丙腈联合小鼠高级腹主动脉瘤建模

The topical elastase murine model of abdominal aortic aneurysm (AAA) is enhanced when combined with &#223;-aminopropionitrile (BAPN)-supplemented drinking ...

advanced-abdominal-aortic-aneurysm-modeling-mice-combination-topical

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Advanced Abdominal Aortic Aneurysm Modeling in Mice by Combination of Topical Elastase and Oral &#223;-aminopropionitrile

1.3K 次观看.  University of Wisconsin-Madison School of Medicine and Public Health. 我们的研究旨在了解腹主动脉瘤的病理生理学。特别是，我们想从分子术语中了解动脉瘤是如何破裂的。我们相信，通过对小鼠、雄性和雌性主动脉瘤进行建模，将帮助我们为这种迫切需要药物治疗的高致死性疾病开发一种新的治疗方法。腹主动脉瘤的病理生理学是多因素和复杂的，这使得动物建模变得困难。然而，弹性蛋白酶/BAPN 模型可靠地...

视频: 作者聚焦:使用 Elastase 模拟人动脉瘤的增强型小鼠 AAA 模型

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase 

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of abdominal aortic aneurysm (AAA) ever since it was first described in rats by Anidjar and colleagues.1 The rationale for its development was based on the disrupted nature of elastin observed in AAAs. This rat model has been modified to produce AAAs in the infrarenal aortic region of mice.2 The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice. Moreover, it is a viable platform to test potential therapeutic agents for AAA. In this video, we demonstrate the elastase infusion AAA procedure used in our laboratory. 
Mice are anesthetized using 2.5% isoflurane, and a laparotomy is performed under sterile conditions. The abdominal aortais isolated with the assistance of an operating stereomicroscope (Leica). After placing temporary ligatures around the proximal and distal aorta, an aortotomy is created at the bifurcation with the tip of a 30-gauge needle. A heat-tapered segment of PE-10 polyethylene tubing is introduced through the aortotomy and secured. The aortic lumen is subsequently perfused for 5-15 minutes at 100 mm Hg with saline containing type I porcine pancreatic elastase (4.5 U/mL; Sigma Chemical Co.). After removing the perfusion catheter, the aortotomy is repaired without constriction of the lumen.

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

This video shows how to induce abdominal aortic aneurysms (AAA) in mice via transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta. The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice.

创建小鼠实验与弹性蛋白酶的腹主动脉瘤

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of ...

科研

生物学

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

在小鼠体内的横向主动脉缩窄

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

医学

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. An aneurysm forms as a result of progressive weakening and eventual dilation of the aorta, which can rupture or tear once it reaches a critical diameter. Aneurysms of the descending aorta in the chest, called descending thoracic aortic aneurysms (dTAA), make up a large proportion of aneurysm cases in the United States. Uncontained dTAA rupture is almost universally lethal, and elective repair has a high rate of morbidity and mortality. The purpose of our model is to study dTAA specifically, to elucidate the pathophysiology of dTAA and to search for molecular targets to halt the growth or reduce the size of dTAA. By having a murine model to study thoracic pathology precisely, targeted therapies can be developed to specifically test dTAA. The method is based on the placement of porcine pancreatic elastase (PPE) directly on the outer murine aortic wall after surgical exposure. This creates a destructive and inflammatory reaction, which weakens the aortic wall and allows for aneurysm formation over weeks to months. Though murine models possess limitations, our dTAA model produces robust aneurysms of predictable size. Furthermore, this model can be used to test genetic and pharmaceutical targets which may arrest dTAA growth or prevent rupture. In human patients, interventions such as these could help avoid aneurysm rupture, and difficult surgical intervention.

We describe a surgical protocol to consistently induce robust descending thoracic aortic aneurysms in mice. The procedure involves left thoracotomy, thoracic aorta exposure, and placement of a sponge soaked in porcine pancreatic elastase on the aortic wall.

局部性乳酸酶诱导下降胸腔主动脉瘤的毛尿外科模型

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. ...

Porcine Model of Infrarenal Abdominal Aortic Aneurysm

Large animal models to study abdominal aortic aneurysms are sparse. The purpose of this model is to create reproducible, clinically significant infrarenal abdominal aortic aneurysms (AAA) in swine. To achieve this, we use a combination of balloon angioplasty, elastase and collagenase, and a lysyl oxidase inhibitor, called &#946;-aminopropionitrile (BAPN), to create clinically significant infrarenal aortic aneurysms, analogous to human disease.
Noncastrated male swine are fed BAPN for 7 days prior to surgery to achieve a steady state in the blood. A midline laparotomy is performed and the infrarenal aorta is circumferentially dissected. An initial measurement is recorded prior to aneurysm induction with a combination of balloon angioplasty, elastase (500 units)/collagenase (8000 units) perfusion, and topical elastase application. Swine are fed BAPN daily until terminal procedure on either postoperative day 7, 14, or 28, at which time the aneurysm is measured, and tissue procured. BAPN + surgery pigs are compared to pigs that underwent surgery alone.
Swine treated with BAPN and surgery had a mean aortic dilation of 89.9% &#177; 47.4% at day 7, 105.4% &#177; 58.1% at day 14, and 113.5% &#177; 30.2% at day 28. Pigs treated with surgery alone had significantly smaller aneurysms compared to BAPN + surgery animals at day 28 (p &#60; 0.0003). The BAPN + surgery group had macroscopic and immunohistochemical evidence of end stage aneurysmal disease.
Clinically significant infrarenal AAA can be induced using balloon angioplasty, elastase/collagenase perfusion and topical application, supplemented with oral BAPN. This model creates large, clinically significant AAA with hallmarks of human disease. This has important implications for the elucidation of AAA pathogenesis and testing of novel therapies and devices for the treatment of AAA. Limitations of the model include variation in BAPN ingested by swine, quality of elastase perfusion, and cost of BAPN.&#160;

This novel model creates robust infrarenal abdominal aortic aneurysms in swine using a combination of balloon angioplasty, elastase/collagenase perfusion, topical elastase application, and oral compound &#946;-aminopropionitrile administration, which interferes with collagen cross-linking.

肾上腺腹动脉瘤的猪模型

Large animal models to study abdominal aortic aneurysms are sparse. The purpose of this model is to create reproducible, clinically significant infrarenal ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

<meta charset="utf8"></head><body>研究小鼠腹主动脉瘤的分子机制

A Mouse Abdominal Aortic Aneurysm Model by Periadventitial Calcium Chloride and Elastase Infiltration

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the abdominal aorta with at least a 50% increase in arterial diameter. Various animal models of AAA have been introduced to mimic the pathophysiological changes and study the underlying mechanisms of AAA. Among these models, the calcium chloride (CaCl2)- and elastase-induced AAA models are commonly used in mice. However, these methods have certain limitations. Traditional intraluminal porcine pancreatic elastase (PPE) perfusion is associated with high technical difficulty and a high rupture rate, while periadventitial administration of PPE yields inconsistent results. In addition, the CaCl2-induced AAA model lacks human AAA features, such as atherothrombosis and aneurysm rupture. Therefore, the combined application of CaCl2 and PPE has been proposed as an approach to enhance success rates and induce greater diameter increases in AAA animal models. This manuscript presents a comprehensive protocol for establishing a mouse AAA model through periaortic infiltration of PPE and CaCl2 in the infrarenal segment of the abdominal aorta. By following this protocol, we can achieve an AAA formation rate of approximately 90% with technical simplicity and reproducibility. Further ultrasound and histological experiments confirm that this model effectively replicates the morphological and pathological changes observed in human AAA.

<meta charset="utf8"></head><body>作者聚焦：腹主动脉瘤小鼠模型的开发和表征

This manuscript presents a protocol for establishing a mouse abdominal aortic aneurysm model using calcium chloride and elastase, combining the advantages of previous modeling methods. This model can be utilized to investigate the pathophysiological mechanisms underlying abdominal aortic aneurysms.

通过外膜周围氯化钙和弹性蛋白酶浸润的小鼠腹主动脉瘤模型

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the ...

局部弹性蛋白酶和口服 β-氨基丙腈联合小鼠高级腹主动脉瘤建模

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此视频中的章节

创建小鼠实验与弹性蛋白酶的腹主动脉瘤

在小鼠体内的横向主动脉缩窄

全基因组基因缺失

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

局部性乳酸酶诱导下降胸腔主动脉瘤的毛尿外科模型

肾上腺腹动脉瘤的猪模型

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

通过外膜周围氯化钙和弹性蛋白酶浸润的小鼠腹主动脉瘤模型

通过外膜周围氯化钙和弹性蛋白酶浸润的小鼠腹主动脉瘤模型