作者想感谢他的技术援助和照顾动物，一个优秀的本科生科研的学生在我们的实验室，安德鲁J唐恩。国家卫生部授予HD058834和密苏里州生命科学奖学金的大学研究所资助的这项工作。

手术前的检测仪器设备/材料<ul><li>剪/钳</li><li>纱布2 × 2“垫</li><li>加热垫</li><li>硝酸银棒</li><li>无菌手术手套</li><li>异氟醚吸入麻醉</li><li>洗必泰葡萄糖4％（防腐剂手术擦洗） </li><li> 2-0无菌手术钢缝合</li><li> 25 × 5 / 8一个针头（蒸压） </li><li>止血钳（蒸压） </li><li>手术钢缝合</li></ul>手术过程<ol><li>创建一个无菌区。按照无菌程序。 </li><li>麻醉鼠标与2 - 3％异氟醚。 </li><li>放置在加热垫的鼠标。 </li><li>洗必泰溶液擦洗尾部。 </li><li>找到5，6，或7 个椎间隙（尾巴骨间颠簸，开始计数的尾巴基地，体毛）。 </li><li>使用止血针和25G两个椎体之间的一个先导孔，到椎间隙。 </li><li>使用钢丝钳削减钢铁缝合10厘米长。 </li><li>取出针和通孔，5厘米的缝合或者尾部插入钢缝合。 </li><li>尾部的每一方应缝合+ 5厘米。 </li><li>放置到拇指尖尾。 </li><li>使用免费的手，风钢缝合到你的拇指尖和扭曲的两端，形成一个大循环。伤口或圆形部分应该是+ 3毫米长。 </li><li>卸下你的拇指。 </li><li>的止血钳缝合链，它弯曲成一个较小的循环，到底这是附尽可能接近原大循环扭曲。 </li><li>切断多余的导线，双绞线和弯曲。 </li><li>如果有任何多余的出血，你可以停止它与硝酸银棒。 </li><li>在一个房子笼恢复5-7天，前后肢卸载，关闭异氟醚和地点的鼠标。如果多个鼠标是住在一起的尾环纠缠。 </li></ol> 1。后肢卸载程序材料<ul><li>大小10-24 × 4机螺钉，每头动物之一。 </li><li>尼龙锁紧螺母大小为10 - 24日，每头动物之一。 </li><li>蝶形螺母大小为10 × 24，每头牲畜。 </li><li>洗衣机大小：× 1，每头牲畜。 </li><li>钓鱼旋转钩大小＃6 1-11/16“，并每动物。 </li><li>金属缝纫机梭芯。每个动物。 </li><li> S形钩的小回形针，每只动物之一</li><li>钳子。 </li><li> 2 × 2“纱布。 </li><li> 2“黑纸夹子。 </li><li>胶带/实验室磁带。 </li><li> 1-3/8“镀槽钢筋笼全长略微超出延伸</li><li> 50厘米x 40CM x 20厘米笼（鼠笼）。 </li><li>弯曲管水壶。 </li><li>不锈钢丝，U形，以支持一瓶水。 </li></ul>网箱设置<ol><li>莱1-38“在笼顶部镀开槽钢筋，和磁带，用胶带暂时下降。 </li><li> 1蝶形螺母，螺杆机螺钉，插入一个垫圈，插入机螺钉槽，开槽的钢板± 15厘米的笼子里的边缘。 </li><li>添加另一洗衣机和蝶形螺母。两个蝶形螺母拧紧，但不要完全拧紧，因为这将有必要做调整后。 </li><li>通过骨架的中心孔插入机螺钉。 </li><li>为了防止脱落骨架，螺丝机螺杆年底尼龙锁紧螺母。骨架应自由旋转。 </li><li>通过下部的骨架，从捕鱼旋转插入钩。 </li><li>用钳子将形成一个S形钩子，从曲别针。 （切2厘米回形针形成一个钩子）。 </li><li>通过渔业旋转孔插入的S形钩子的一端，用钳子捏它关闭。 </li><li>将U形线，内笼边平行的机螺钉，弯曲两端支持一个完整的一瓶水。 </li><li>插入U形线的一瓶水，spicket面临下降，但不触及底部。 </li><li>弯曲的2“粘合剂钳开放，因此，他们可以不关闭关闭。只要足够的张力以持有它没有它下跌了3到4件的食品，防止破碎的食品钳的力量，或取缔鼠标的鼻子。 </li><li>放置3至4件灭鼠议员进入粘结剂钳。 </li></ol>小鼠后肢卸载<ol><li>通过较小的戒指尾戒插入开口端的S形钩子。 </li><li>调整鼠标的高度较低的蝶形螺母，机器上的螺丝拧紧或松动。下肢不能负重。一旦实现最佳的高度是拧紧上部的蝶形螺母。 </li><li>一瓶水，和U形线重新定位，使鼠标可以到spicket攀升，其后肢。<html>安全的矿泉水瓶用胶带笼的一侧。 </li><li>笼底的食物的粘结剂钳的位置，使鼠标无法爬上磁带，用胶带。 </li><li>重复相同的步骤，如果有一个以上的动物是后肢笼子的另一侧卸。众议院两个动物，每笼空间，使他们几乎可以触摸对方的动物。 </li><li>使用2 × 2纱布，以松散的包装上的鱼旋转鼠标的尾部。使用的磁带的小片环绕的纱布，纱布尾巴。这一步很重要，以防止下垂向下的尾巴，导致血液汇集在尾部的头部和尾部成为坏死。 </li><li>添加少量的COB床上用品笼子。不要添加太多的铺垫，否则鼠标从一个土堆的床上用品和床上用品尝试加载后肢。 </li></ol> 2。代表性的成果： 要建立尾胡环技术的成功，我们测量动物体重尾戒手术前，在此之前，胡锦涛和恢复;比目鱼肌质量;在所有这些群体肾上腺肿块。 如图1所示，整个实验的老鼠的体重。从尾部环手术（24.3 ±2.9克）之前胡复苏后5天尾戒手术（24.7 ± 3.5G）前​​的小鼠的体重没有显着差异。体重也没有明显下降后，立即胡锦涛14天（22.7 ±1.9克），28日，胡天（21.3 ±2.1克）或后3天胡（24.0 ±1.8克）恢复。虽然胡14和28天在身体质量小（-6.8％和12％），但不显着的损失，小鼠完全康复前的胡体重后3天的恢复。卧床对照组小鼠和胡小鼠的食品消费是如图3​​所示。平均每日食物摄入量不超过28天胡小鼠不同，与对照组相比。 我们观察到显着的比目鱼肌萎缩，14日和28日表示一个有效的胡程序（图2）（-39.1％和-46.6％）胡。此外，这种程度的萎缩，预计2-4 胡2，7，8周文献值一致。以下3天胡比目鱼大规模的恢复显着增加至74％的控制值。在恢复组的比目鱼质量明显比胡组，但仍明显高于日间控制动物。 肾上腺重量测定动物应力指标，左，右肾上腺切除和权衡。的平均体重控制和胡小鼠各组小鼠（图4）相媲美。 以前在老鼠我们已经表明，在胡锦涛开始对动物的成功率相比，动物的数量，完成胡不悬挂装置（由于动物通过尾部的牵引带嚼）10 12莫雷霍尔顿技术1。在这项研究中的小鼠（N = 54）有否来自悬挂下来之前，胡期间完成。然而，我们确实有一个鼠标，开发出一种严重发炎的尾巴。当炎症成为明显，我们删除的尾戒，让发炎面积愈合，并随后放在另一尾环胡锦涛完成期限。 在过去我们已经使用了胡莫雷霍尔顿方法时，我们通常消除1-2小鼠/组，因为他们来悬挂装置。随着线索环法有无意中在为期14天或28天（N = 54只小鼠）胡锦涛胡仪器的小鼠没有发作。 <img src="/files/ftp_upload/2467/2467fig1.jpg" alt="图1" /> 图1。体重的变化（平均值± SD）。缩写：缺点：对照组小鼠负重（N = 76）;预胡：胡（N = 54）之前的所有小鼠; HU14：14天（N = 34）小鼠后肢卸载; HU28：小鼠后肢卸载为28天（N = 20）;录音：允许笼卸载小鼠后肢恢复为3天（N = 22）。观察组之间的体重无显着差异。 <img src="/files/ftp_upload/2467/2467fig2.jpg" alt="图2" /> 图2。比目鱼肌萎缩（平均值± SD）。缩写：缺点：负重对照组（N = 76）; HU14：卸载为14天（N = 34）小鼠后肢; HU28：小鼠后肢卸载;为28天（N = 20）和REC：后肢允许卸载小鼠笼中恢复为3天（N = 22）。 *显着性差异（P &lt;0.001），比负重控制（CON）。 δSignificantly不同的两个胡组（P &lt;0.01）。 467fig3.jpg“ALT =”图3“/&gt; 图3。食物消费量（平均值± SD）。平均每日食物摄入量（克/日），在负重和后肢卸载。缩写：负重对照组（N = 76）;胡：小鼠后肢unloaded14 - 28天。 CON和胡组之间无显着性差异。 <img src="/files/ftp_upload/2467/2467fig4.jpg" alt="图4" /> 图4。肾上腺肿块（平均值± SD）。缩写：缺点：负重对照组（N = 76）; HU14：卸载为14天（N = 34）小鼠后肢; HU28：小鼠后肢卸载;为28天（N = 20）和REC：后肢允许卸载小鼠笼中恢复为3天（N = 22）。观察组与组之间无显着差异。

<ol>
	<li>Brown, M., Foley, A. M., Hasser, E. M., Foley, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+method+for+hindlimb+unweighting.">An alternative method for hindlimb unweighting.</a> J Gravitational Physiol. 12, 43-50 (2005).</li><li>Fitts, R. H., Metzger, J. M., Riley, D. A., Unsworth, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+disuse:+a+comparison+of+hindlimb+suspension+and+immobilization.">Models of disuse: a comparison of hindlimb suspension and immobilization.</a> J Appl Physiol. 60, 1946-1953 (1986).</li><li>Ingalls, C. P., Warren, G. L., Armstrong, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Ca2++transients+in+mouse+soleus+muscle+after+hindlimb+unloading+and+reloading.">Intracellular Ca2+ transients in mouse soleus muscle after hindlimb unloading and reloading.</a> Journal of Applied Physiology. 87, 386-390 (1999).</li><li>Morey, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spaceflight+and+bone+turnover:+correlation+with+a+new+rat+model+of+weightlessness.">Spaceflight and bone turnover: correlation with a new rat model of weightlessness.</a> BioScience. 29, 168-172 (1979).</li><li>Morey-Holton, E. R., Globus, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindlimb+unloading+of+growing+rats:+a+model+for+predicting+skeletal+changes+during+space+flight.">Hindlimb unloading of growing rats: a model for predicting skeletal changes during space flight.</a> Bone. 22, 83-88 (1998).</li><li>Morey-Holton, E. R., Globus, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindlimb+unloading+rodent+model:+technical+aspects.">Hindlimb unloading rodent model: technical aspects.</a> J Appl Physiol. 92, 1367-1377 (2002).</li><li>Thomason, D. B., Booth, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrophy+of+the+soleus+muscle+by+hindlimb+unweighting.">Atrophy of the soleus muscle by hindlimb unweighting.</a> J Appl Physiol. 68, 1-12 (1990).</li><li>Thomason, D. B., Herrick, R. E., Surdyka, D., Baldwin, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+soleus+muscle+myosin+expression+during+hindlimb+suspension+and+recovery.">Time course of soleus muscle myosin expression during hindlimb suspension and recovery.</a> J Appl Physiol. 63, 130-137 (1987).</li></ol>

我们评价后肢卸载小鼠的替代方法，我们决心非常成功。此处所述的尾环法是用于长期后肢悬挂，并研究恢复胡。每个这些条件下，没有任何证据未能适应，稳定的食物摄入量，体重，和小鼠肾上腺重量。这个尾环胡技术可以方便地执行，并可以很容易地用非常小的手术实践中。 尾环法有几个优势，相比传统的莫雷霍顿5，6方法。第一，皮肤和环植入网站上，尾巴总是可见的，所以如果出现问题，这是显而易见的立即，而不是仅仅在暂停期间结束。二，动物是无法走出的悬挂装置在任何时间，在胡，省却了需要做出决定是否要保留一个鼠标或数据集。此外，老鼠不打扰环装置，当他们从胡复苏的释放，他们不会乱动他们的尾巴，允许一个无缝的复苏阶段，下面的胡。最后，后肢与尾环法卸载小鼠保持自己的体重比小鼠后肢卸载莫雷霍尔顿尾牵引技术。在我们的手中胡锦涛14天之后，我们观察到在身体质量小（-6.8％），但不显着的损失。鉴于英格尔斯等。后观察14天胡莫雷霍尔顿方法3在在小鼠体重明显下降-12％。虽然英格尔斯没有报告肾上腺重量，看来，我们的尾环法赋予鼠标（在我们的胡小鼠肾上腺重量不超过控制不同），更好地维护整个后肢卸货期间体重更小的压力。 总之，后肢卸尾环法对小鼠胡测试14和28天，对小鼠产生笼恢复为3天之后，胡。小鼠肾上腺重量，体重和食物摄入量的基础上，似乎有尾环设备很好地适应。尾环后肢卸载方法是有用的，简单的替代传统莫雷霍尔顿后肢装卸技术。

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Wire cutters/pliers</td>
<td>&nbsp;</td>
<td>Ace Hardware </td>
<td>84-056</td>
<td>&nbsp;</td>
<tr>
<td>Gauze</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>22-037-901</td>
<td>&nbsp;</td>
<tr>
<td>Heating pad</td>
<td>&nbsp;</td>
<td>Sunbeam</td>
<td>Z-1228-001</td>
<td>&nbsp;</td>
<tr>
<td>Silver nitrate applicators</td>
<td>&nbsp;</td>
<td>ARZOL</td>
<td>0001-2</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane,USP</td>
<td>&nbsp;</td>
<td>Novaplus</td>
<td>NDC 0409-3292-49</td>
<td>&nbsp;</td>
<tr>
<td>Chlorhexidine Gluconate 4%</td>
<td>&nbsp;</td>
<td>Purdue Products L.P</td>
<td>NDC 67618-200-01</td>
<td>&nbsp;</td>
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<td>Sterile 2-0 surgical steel suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>SS-28</td>
<td>&nbsp;</td>
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<td>25 X 5/8 A gauge needle</td>
<td>&nbsp;</td>
<td>Terumo</td>
<td>NN 2516R</td>
<td>&nbsp;</td>
<tr>
<td>Hemostat/ needle holder</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>12500-12</td>
<td>&nbsp;</td>
<tr>
<td>Size 10-24 X 4 machine screws</td>
<td>&nbsp;</td>
<td>Hillman The Fastener Center</td>
<td>90272</td>
<td>&nbsp;</td>
<tr>
<td>Nylon lock nut size 10 X 24</td>
<td>&nbsp;</td>
<td>Hillman The Fastener Center</td>
<td>180141</td>
<td>&nbsp;</td>
<tr>
<td>Wing nut size 10 X 24</td>
<td>&nbsp;</td>
<td>Hillman The Fastener Center</td>
<td>180243</td>
<td>&nbsp;</td>
<tr>
<td>Fender Washer size &frac14; X 1</td>
<td>&nbsp;</td>
<td>Hillman The Fastener Center</td>
<td>290012</td>
<td>&nbsp;</td>
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<td>Fishing swivel with snap hook size #6, 1-11/16&#x201D;</td>
<td>&nbsp;</td>
<td>Bass Pro Shop</td>
<td>38-481-554-01</td>
<td>&nbsp;</td>
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<td>Metal sewing machine bobbin.</td>
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<td>Singer</td>
<td>45785</td>
<td>&nbsp;</td>
<tr>
<td>Paper clip</td>
<td>&nbsp;</td>
<td>Office Depot</td>
<td>221720</td>
<td>&nbsp;</td>
<tr>
<td>2&#x201D; black paper binder clamps.</td>
<td>&nbsp;</td>
<td>Office Depot</td>
<td>308957</td>
<td>&nbsp;</td>
<tr>
<td>Scotch Multi-Use Duct Tape</td>
<td>&nbsp;</td>
<td>Office Depot</td>
<td>790710</td>
<td>&nbsp;</td>
<tr>
<td>1-3/8&#x201D; Plated slotted steel bar</td>
<td>&nbsp;</td>
<td>Home Depot</td>
<td>548932</td>
<td>Extends slightly beyond the full length of the cage</td>
</tr>
<tr>
<td>50cm X 40cm X 20cm cage (rat cage</td>
<td>&nbsp;</td>
<td>Alternative Design Manufacturing</td>
<td>RC88D-PC</td>
<td>&nbsp;</td>
<tr>
<td>Water Bottle</td>
<td>&nbsp;</td>
<td>Alternative Design Manufacturing</td>
<td>WB16FS</td>
<td>&nbsp;</td>
<tr>
<td>Stainless Steel wire</td>
<td>&nbsp;</td>
<td>Small Parts, Inc</td>
<td>SWX-4029</td>
<td>U-shaped wire to support water bottle</td>
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an alternant method to the traditional nasa hindlimb unloading model in mice

莫雷霍尔顿后肢卸载（胡）方法是一种被广泛接受的美国国家航空和航天局（NASA）地面模型为基础的研究在啮齿类动物中的废用4-6萎缩。我们的研究评估的金标准莫雷霍尔顿胡小鼠尾牵引技术交替的方法。五十四雌性小鼠（4-8谟。）胡锦涛为14天（N = 34）或28天（N = 20）。从胡的恢复评估后3天的正常笼下地胡（N = 22）。现年匹配的小鼠（N = 76）担任负重控制。 胡锦涛之前尾巴环形成了2-0无菌手术的钢丝，通过5日，6日或7日间的椎间盘空间，并塑造成一个环，从小鼠暂停。椎尾环的位置选择适当平衡，而不会干扰与排便动物体重。 我们确定了这本小说的胡技术评估之前和之后，比目鱼肌萎缩的程度，胡和肾上腺肿块胡的体重成功。小鼠的身体的重量之前，胡（24.3 ±2.9克）没有显着下降之后，14D（22.7 ±1.9克）胡，胡28D（21.3 +2.1克）或3天痊愈后立即（24.0 ±1.8克） 。比目鱼肌质量显着下降（-39.1％和-46.6％），胡分别为14天和28天（P &lt;0.001）。以下3天恢复比目鱼质量显着提高到74％的控制值。胡小鼠肾上腺重量差异不显着，与对照组小鼠相比，。 胡相应的文献值预计，2，7，8的动物体重，肾上腺重量可比，和比目鱼肌萎缩的维护我们的新颖胡方法的成功就是明证。本胡法的主要优点包括：1）缓解尾考试期间暂停; 2）经常与观察尾部录音和胡紫绀，发炎，和/或坏死的尾巴的可能性下降; 3）没有对小鼠的可能性咀嚼的牵引磁带和悬挂装置，以及4）迅速恢复和正常的活动后，立即胡笼。

Watch this Scientific Journal Video about An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice at JoVE.com

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

我们开发了一个交替后肢卸在小鼠模型。后肢卸尾环比传统莫雷-霍尔顿尾牵引技术方法的主要优点是简单直白的过程，最大限度地减少动物时的应力。

传统美国宇航局后肢卸小鼠模型的一个交替的方法

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

an-alternant-method-to-traditional-nasa-hindlimb-unloading-model

Research

JoVE Journal

Biology

20.1K 次观看.  University of Missouri, Columbia. 我们开发了一个交替后肢卸在小鼠模型。后肢卸尾环比传统莫雷-霍尔顿尾牵引技术方法的主要优点是简单直白的过程，最大限度地减少动物时的应力。

视频: An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

肢体缺血小鼠模型

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

科研

生物学

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

混合式切割：用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

评估细菌素对小鼠肠道微生物的影响的方法

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

从小鼠中分离心肌细胞的安特格拉德灌注方法

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

通过粘附足迹测定法对细胞滚动中的分子粘附进行成像

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

小鼠自身免疫性糖尿病过继转移模型中移植物存活的生物发光监测

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

建立小鼠骨干股骨骨折模型

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...