这项工作得到了瑞士达沃斯 RISystem 股份公司的支持。

所有的程序都是根据国家卫生研究院关于使用实验动物的指导方针进行的, 并遵循机构指南 (Landesamt Verbraucherschutz、Zentralstelle Amtstierärztlicher Dienst、萨尔布吕肯、德国)。
1. 外科器械和植入物的制备
<ol><li>选择手术刀刀片 (大小 15), 小拭子, 细钳, 27 克针, 非可吸收5-0 缝合, 剪刀和针持有人从显微外科仪器盒。</li>
	<li>解锁髓内螺钉、导丝 (直径 0.3/0.2 毫米、10厘米长)、定心钻头 (0.5 毫米直径) 和手钻 (图 1; 请参阅材料表)。 注: 髓内螺钉 (0.5 毫米直径, 17.2 毫米长度) 是由医疗级不锈钢制成, 用于逆行植入股骨。该螺钉有一个近端螺纹 (直径0.5 毫米, 4 毫米长度) 与鼻子 (0.2 毫米直径, 0.4 毫米长度) 在尖端和远端锥形头 (0.8 毫米直径, 0.9 毫米长度), 以实现骨折压缩以及轴向和旋转稳定性。</li>
	<li>将植入物和所有手术器械暴露在消毒液中 (96% 酒精) 5 分钟或消毒 (蒸汽灭菌, 130 摄氏度, 25 分钟)。消毒或灭菌后, 将仪器放置在操作布上。将操作布直接放置在小动物操作桌旁。</li>
</ol>2. 动物、麻醉和镇痛
<ol><li>根据所讨论的研究问题, 选择小鼠的应变、年龄和性别。 注: 本研究使用 12-至14周的雄性 CD-1 小鼠。适当的体重使用髓内螺钉是在25-35 克之间。</li>
	<li>麻醉小鼠腹腔注射15毫克/千克甲苯噻嗪和75毫克/千克氯胺酮。通过脚趾夹来确认麻醉。使用眼部润滑剂, 以保护动物的眼睛在麻醉期间的干燥。麻醉后, 将鼠标置于散热器下, 保持体温恒定。在手术过程中, 动物受到反复脚趾捏的监测, 以确保适当的麻醉面。</li>
	<li>应用盐酸曲马多在饮用水 (1.0 毫克/毫升) 镇痛从1天之前的手术, 直到3天手术后。 注: 镇痛和感染预防应符合国家和机构在进行实验的相关指导方针。</li>
</ol>3. 外科手术和髓内螺钉植入术
<ol><li>手术前, 剃掉整条右后腿, 涂上脱毛膏。5分钟后, 取出奶油, 用清水清洁腿部。然后, 使用96% 酒精消毒液。Betadine 或洗必泰可以添加到酒精, 以确保完全无菌。</li>
	<li>在无菌条件下, 将鼠标放在小动物操作台上的仰卧位上。弯曲右膝, 允许前路髁膝。使用手术刀刀片在右膝上执行5毫米内侧 parapatellar 切口。</li>
	<li>用手术刀刀片和拭子仔细地调动髌骨韧带。然后, 用细钳将髌骨侧面移位, 以暴露股骨髁间凹槽。</li>
	<li>在两个髁之间, 在股骨中部完全打开髁间凹槽。请确保钻孔的深度不要超过1.0 毫米。<ol>
<li>使用 0.5 mm 定心钻头和手钻 (图 1C和D,图 2), 以慢速和45°偏移腹部到股骨轴进行手动钻孔。在钻探过程中, 持续减少角度到0°偏移 (平行与骨轴的股骨)。当达到1.0 毫米深度时, 停止钻孔。</li>
	</ol>
</li>
	<li>在髁间凹槽处打开骨后, 将27克针插入髓内腔, 使其在股骨的全长。通过27克针的旋转运动, 手动将股骨髓内腔固定。向前推针, 以穿孔的皮质骨在大转子下部。</li>
	<li>取出27克针, 并将导丝通过股骨远端。<ol>
<li>用手术刀刀片 (尺寸 15) 在导丝上进行皮肤切口, 并将导丝向前推, 直到导丝两端都在外面。请务必把导丝放到位。</li>
	</ol>
</li>
	<li>使用断头台创建定义闭合的断裂。<ol>
<li>将鼠标放在断头台下的右腿位置。确保股骨的骨干部分放在断头台的中间。</li>
		<li>将重量 (200 克) 从所定义的25.5 厘米的距离中除去。</li>
	</ol>
</li>
	<li>使用 x 射线设备控制断开的配置和断开位置以及导丝 (图 3) 的位置 (请参阅材料表)。</li>
	<li>将髓内螺钉与远端的鼻部连接至0.2 毫米导丝, 并在连续压力下将其插入股骨, 并顺时针旋转。<ol>
<li>当达到足够扭矩时, 驱动轴的剪切。</li>
		<li>卸下导丝下部。</li>
	</ol>
</li>
	<li>用5-0 合成、单丝、美容整形聚丙烯缝合术, 将髌骨重新定位, 用一条缝合固定髌骨肌腱。使用相同的材料和大小的单一缝合闭合伤口。使用 x 射线设备控制碎片和螺钉位置放射学上的减少 (请参阅材料表)。</li>
	<li>把动物放在散热器下, 直到他们从麻醉中恢复。不要离开动物无人看管, 直到他们恢复足够的意识, 以保持腹卧床。把动物送到动物设施的单人笼子里。在头24小时内, 不要将动物送回其他动物的公司, 即使他们已经完全从麻醉中恢复过来。</li>
	<li>每天仔细监视动物。在头三天内用1.0 毫克/毫升的饮用水维持术后镇痛。继续镇痛如果, 在手术后的4天, 动物仍然显示疼痛的证据, 如发声, 躁动, 缺乏机动性, 没有新郎, 异常的姿势, 和缺乏正常的环境兴趣。当动物无痛时, 终止镇痛。</li>
	<li>实验结束时, 弄死的巴比妥过量。</li>
</ol>

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作者声明他们没有竞争的财政利益。

手术步骤的关键步骤是找到正确的入口点的螺钉植入在股骨中部髁在髁间凹槽, 以及最佳方向平行的针骨轴为扩孔髓内腔。为了避免输入位置不正确, 外科医生应该准备好切口, 直到达到最佳的视野。为了控制扩孔过程中的定位, 应将小鼠的股骨固定在手指的稳定位置。另一个关键步骤是将螺钉插入到股骨上方的导丝上, 因为导丝可以从近端骨碎片中滑出, 导致骨折脱位。在这种情况下, 外科医生可以尝试再次线程的骨头碎片, 但这一动作大多是不成功的, 和动物, 然后被排除在研究。
此外, 手术过程可能会产生一些并发症。例如, 髌骨韧带, 这是横向转移, 以获得最佳的看法, 髁, 可能会破裂。这需要在螺钉插入后缝合韧带。在打开髁骨和扩髓内腔时, 髁可能爆裂。在这种情况下, 不存在故障排除的可能, 因为螺钉在远端无法充分固定, 并且无法实现对骨折的压缩。另一种并发症是插入的导丝的脱位或骨不正确的位置。这种并发症可以通过谨慎处理和植入后的影像分析来减少, 以确认手术中正确的放置。此外, 外科医生应该注意螺钉完全插入, 因为螺钉的凸出可以限制鼠标的移动或减少骨折的压缩。因此, 一个 x 射线设备是强制性的外科手术。只有在手术结束后的影像证实的动物应纳入研究协议。
在实验结束时将髓内螺钉取出, 可以不碰到任何困难, 因为螺钉的头可以连接到特殊的移除仪上, 或者, 螺钉也可以与针架一起移除。
该技术的一个局限性是, 该公司提供的髓内螺钉只有一个尺寸, 长度为17.2 毫米, 因此必须考虑股骨的大小。对髓内螺钉使用的进一步限制是,体内微计算机断层扫描 (CT) 或磁共振成像 (MRI) 分析的愈合过程几乎是不可能的, 由于植入材料, 影响图像质量.因此, 这些分析只能在安乐死后进行, 并在研究期结束后摘除植入物。最后, 螺钉不能用于分析骨缺损愈合, 因为轴向稳定性是通过对骨碎片的近端螺纹和远端头部的压缩实现的。
骨骼愈合研究使用开放的8,9,10,11,1213,14或关闭4,15, 16,17骨骼愈合模型。开放性骨愈合模型允许更硬的片段固定与闭合骨愈合模型相比, 导致更高的膜内愈合量没有明显的愈伤组织形成。由于开放模型与小愈伤组织的形成有关, 这些模型可能不可取的实验, 需要大量的愈伤组织的生物化学和分子分析。开放模型的一个进一步缺点是需要一个侵入性侧向方法与主要软组织创伤。相比之下, 闭合模型的使用只需要一个较小的侵入性切口。直到现在只有几个闭合的模型在老鼠2中存在。
在闭合的骨头愈合模型, 主要使用一个简单的髓内针。然而, 这种技术有明显的缺点。最明显的是, 缺乏轴向和旋转的稳定性。这可能导致异类愈合响应5。虽然已知这一缺点影响实验结果18, 最近的研究, 打算分析骨骼愈合的机制, 仍然使用小鼠模型, 其中骨折是稳定的只有一个 pin 或甚至左 unstabilized7.我们认为, 稳定的接骨术技术, 可与临床实践中使用的方法相媲美, 也应用于小鼠。为实现轴向和旋转稳定性, 研制了髓内螺钉, 通过远端头和近端螺纹诱导骨折的压缩。有兴趣的是, 髓内螺钉的应用不产生刚性固定, 因此, 与骨折股骨相比, 带髓内螺钉稳定的骨折股骨的扭转刚度明显降低。由外固定架或锁定板5稳定。然而, 要研究软骨骨愈合, 需要一种不太僵硬的固定方法, 因为只有较不刚性的固定, 才能使 micromovements 骨碎片, 从而引发软骨愈合过程。然而, 如前一例活体研究所示, 髓内螺钉产生明显的轴向和旋转稳定性。生物力学分析显示, 髓内螺钉的扭转刚度为 0.34 0.18 Nmm/°, 与传统引脚 (0.00 @ 0.00 Nmm/°)5相比, 这一结果显著提高。因此, 这里介绍的髓内螺钉是唯一的植入物, 可用于微创技术和提供标准化的生物力学条件的研究软骨骨折愈合的小鼠。

由于广泛的抗体和基因改良的动物, 小鼠的骨愈合研究是非常需要的。这些事实允许研究骨骼愈合的分子机制1。在过去的几年中, 不同的骨骼愈合模型的小鼠已经开发了2。这些模型可以分为开放模型, 其中骨骼是 osteotomized 使用开放的外侧手术方法和封闭模型, 其中骨是骨折的基础上, 由 Bonnares 和埃霍恩介绍的断裂模型3。使用这种技术, 一个标准化的横向骨折可以由一个3点弯曲装置和髓内植入物可以插入通过一个小的内侧 parapatellar 切口的微创技术, 以避免重大软组织创伤。
髓内螺钉可用于小鼠闭合性骨折的稳定。螺杆提供旋转和轴向稳定性。这是通过断开压缩通过近端线程和远端头4实现的。螺钉的进一步优点是手术技术简单, invasivity 低, 种植体重量低, 最显著的是, 与其他髓内固定相比, 提供标准化和可控制的生物力学条件的稳定性更高。植入5。事实上, 在最闭合的断裂模型中, 碎片只通过简单的针脚稳定, 这与完全缺乏旋转和轴向稳定性以及高的针脚风险和断裂脱位有关。这会明显影响愈合过程, 这可能导致延迟愈合或非联合形成。
众所周知, 骨折固定的稳定性对愈合过程有巨大影响6,7。高刚性固定的结果膜内愈合, 而不刚性固定, 这可能允许 micromovements 在骨折间隙, 导致软骨愈合。骨折与髓内螺钉的稳定主要表现为软骨愈合与大量的愈伤组织, 特别是经过2周的骨折愈合。收获大量愈伤组织的可能性可以通过不同的技术分析多种参数。
本文报道了小鼠髓内螺钉的设计和应用, 以及在正常软骨骨愈合实验研究中的优缺点。

<table>
	<tbody>
		<tr>
			<td>Mouse Screw</td>
			<td>RISystem AG</td>
			<td>221,100</td>
			<td></td>
		</tr>
		<tr>
			<td>Guide wire</td>
			<td>RISystem AG</td>
			<td>521,100</td>
			<td></td>
		</tr>
		<tr>
			<td>Centering bit</td>
			<td>RISystem AG</td>
			<td>590,205</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand drill</td>
			<td>RISystem AG</td>
			<td>390,130</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton-Swab (150 mm, small head)</td>
			<td>Fink Walter GmbH</td>
			<td>8822428</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture (5-0 Prolene)</td>
			<td>Ethicon</td>
			<td>8614H</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Braun Aesculap AG &#38;CoKG</td>
			<td>BD520R</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Braun Aesculap AG &#38;CoKG</td>
			<td>BC100R</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Braun Aesculap AG &#38;CoKG</td>
			<td>BM024R</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G needle</td>
			<td>Braun Melsungen AG</td>
			<td>9186182</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade size 15</td>
			<td>Braun Aesculap AG &#38;CoKG</td>
			<td>16600525</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat radiator</td>
			<td>Sanitas</td>
			<td>605.25</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Asid bonz GmbH</td>
			<td>NDXZ10</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye lubricant</td>
			<td>Bayer Vital GmbH</td>
			<td>2182442</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer Vital GmbH</td>
			<td>1320422</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Serumwerke Bernburg</td>
			<td>7005294</td>
			<td></td>
		</tr>
		<tr>
			<td>Tramadol</td>
			<td>Gr&#252;nenthal GmbH</td>
			<td>2256241</td>
			<td></td>
		</tr>
		<tr>
			<td>Disinfection solution (SoftaseptN)</td>
			<td>Braun Melsungen AG</td>
			<td>8505018</td>
			<td></td>
		</tr>
		<tr>
			<td>CD-1 mice</td>
			<td>Charles River</td>
			<td>22</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray Device</td>
			<td>Faxitron MX-20, Faxitron X-ray Corporation</td>
			<td>2321A0988</td>
			<td></td>
		</tr>
		<tr>
			<td>Fracture device small</td>
			<td>RISystem AG</td>
			<td>891,100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a minimally invasive model to analyze endochondral fracture healing in mice under standardized biomechanical conditions

骨愈合模型是必要的分析骨折愈合的复杂机制, 以改善临床骨折治疗。在过去的十年中, 在骨科研究中增加了老鼠模型的使用, 这很可能是因为老鼠模型提供了大量的基因改良菌株和特殊的抗体来分析骨折愈合的分子机制。为了控制生物力学条件, 具有良好特征的内固定技术是强制性的, 也在小鼠。在这里, 我们报告的设计和使用闭合骨愈合模型, 以稳定股骨骨折的小鼠。采用医用不锈钢制成的髓内螺钉, 通过与主要使用的简单髓内针相比, 可提供轴向和旋转稳定性, 这表明完全缺乏轴向和旋转稳定性。用髓内螺钉实现的稳定性可以分析软骨愈合。大量的愈伤组织, 在与螺钉稳定后接受, 提供了理想的条件, 以收获组织的生物化学和分子分析。螺钉使用的另一个好处是, 螺钉可以用微创技术插入股骨, 而不会引起软组织损伤。总之, 螺钉是一种独特的植入物, 最好能用于闭合性骨折愈合模型, 提供标准化的生物力学条件。

从皮肤切口到伤口闭合的手术时间为20分钟。手术可以在没有立体显微镜的情况下进行。术后每天对动物进行监测。术后镇痛在2天后终止, 因为没有动物在这个时间段后显示疼痛的证据。在手术后2天内, 动物也显示正常的负重。在整个观察期内未观察到伤口感染。
2周后的放射性分析表明, 愈伤组织的形成, 弥合骨折间隙 (图 4A)。5周后, 骨折愈合, 骨膜愈伤组织几乎完全改建 (图 4B)。
2周后愈伤组织和骨折区的组织学分析表明, 在软骨过程和梭织骨中建立的软骨组织软骨愈合的典型组织分布 (图 5A)。5周后, 软骨组织消失, 梭织骨被转换成片状骨, 以重建骨骼的正常解剖和承载特性 (图 5B)。
2周后的生物力学分析表明, 与对侧断开骨相比, 弯曲刚度为37%。5周后, 弯曲刚度几乎为 100%, 表示完全愈合 (图 6)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57255/57255fig1.jpg"> 图 1: 植入.A.髓内螺钉 (直径0.5 毫米, 17.2 毫米长度), 螺纹 (0.5 毫米直径, 4 毫米长度) 和鼻子 (0.2 毫米直径, 0.4 毫米长度) 近端和圆锥状头 (0.8 毫米直径, 0.9 毫米长度) 远端。B.导丝 (直径 0.3/0.2 毫米, 10 厘米长)。C. 中心钻头 (直径0.5 毫米)。D.手钻。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57255/57255fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57255/57255fig2.jpg"> 图 2: 股骨髁的示意图, 指示髓内螺钉的入口点.股骨髁的髁间凹槽在前后视 (左) 和矢状视图 (右)。十字 (左) 表示髓内螺钉的入口点, 箭头 (右) 表示要开始钻孔的股骨轴的45°偏移量。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57255/57255fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57255/57255fig3.jpg"> 图 3: 带横向骨折的股骨的射线照相和插入的导丝.x 线显示股骨 (箭头) 骨干部分的横向骨折结构, 以及髓内腔内连接骨折的导丝。刻度条表示5毫米.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57255/57255fig3large.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/57255/57255fig4.jpg"> 图 4: 2 和5周的骨骼愈合后的射线照相.a. 2 周后用螺钉稳定的股骨的影像学分析, 显示明显的愈伤组织形成。B. 5 周后用螺钉固定的股骨的影像学分析, 表明骨折愈合与愈伤组织的重建几乎完全相同。刻度条表示5毫米.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57255/57255fig4large.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/57255/57255fig5.jpg"> 图 5: 在骨骼愈合2和5周后组织学切片.a. 2 周后用螺钉稳定股骨的组织学分析, 表明软骨骨愈合过程中的典型组织分布 (c) 及愈伤组织内的骨骼 (b)。B.在5周后用螺钉稳定股骨的组织学分析, 表明对板层骨的几乎完全重塑。组织学切片根据三色方法染色。刻度条代表1000µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57255/57255fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57255/57255fig6.jpg"> 图 6: 生物力学分析.2周后弯曲刚度的生物力学分析 (白条、n=9) 和5周 (黑条、n=8)。弯曲刚度的百分比给对侧非骨折股骨。数据作为平均值的标准误差 (SEM), * p < 0.05 vs 2 周。在证明了正态分布 (柯尔莫哥洛夫斯米尔诺夫试验) 和等方差 (F 检验) 假设后, 采用 Student´s t 检验法对两组实验进行了比较。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57255/57255fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions at JoVE.com

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

本协议描述了一种微创内固定技术, 采用髓内螺钉规范化稳定股骨骨折, 可用于分析软骨骨愈合的小鼠。

标准化生物力学条件下小鼠软骨骨折愈合的微创模型分析

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

a-minimally-invasive-model-to-analyze-endochondral-fracture-healing

Nanyang Technological University

Research

JoVE Journal

Medicine

8.2K 次观看.  Saarland University. 本协议描述了一种微创内固定技术, 采用髓内螺钉规范化稳定股骨骨折, 可用于分析软骨骨愈合的小鼠。

视频: A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

科研

医学

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

光化学缺血:一种微创和可重复性的光化学皮质病变模型小鼠中风研究

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

胫骨神经切断术 - 一个标准化的模型失神经诱导小鼠骨骼肌

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

髓内锁钉股骨截骨标准化固定用于分析正常和有缺陷的骨愈合小鼠

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

Minimally Invasive Transverse Aortic Constriction in Mice

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard open-chest transverse aortic constriction (TAC). Although transverse aortic constriction is a highly functional method for the induction of high pressure in the left ventricle, it is a more difficult and lengthy procedure due to its use of artificial ventilation with tracheal intubation. TAC is oftentimes also less survivable, as the newer method, MTAC, neither requires the cutting of the ribs and intercostal muscles nor tracheal intubation with a ventilation setup. In MTAC, as opposed to a thoracotomy to access to the chest cavity, the aortic arch is reached through a midline incision in the anterior neck. The thyroid is pulled back to reveal the sternal notch. The sternum is subsequently cut down to the second rib level, and the aortic arch is reached simply by separating the connective tissues and thymus. From there, a suture can be wrapped around the arch and tied with a spacer, and then the sternal cut and skin can be closed. MTAC is a much faster and less invasive way to induce left ventricular hypertension and enables the possibility for high-throughput studies. The success of the constriction can be verified using high-frequency trans-thoracic echocardiography, particularly color Doppler and pulsed-wave Doppler, to determine the flow velocities of the aortic arch and left and right carotid arteries, the dimension of the blood vessels, and the left ventricular function and morphology. A successful constriction will also trigger significant histopathological changes, such as cardiac muscle cell hypertrophy with interstitial and perivascular fibrosis. Here, the procedure of MTAC is described, demonstrating how the resulting flow changes in the carotid arteries can be examined with echocardiography, gross morphology, and histopathological changes in the heart.

Minimally invasive transverse aortic constriction (MTAC) conserves the essentials of regular transverse aortic constriction (TAC) while eliminating the use of a ventilator with tracheal intubation. It proves to be a highly desirable method for high-throughput studies on left ventricular overload, particularly in translational studies.

微创横向主动脉缩窄小鼠

Minimally invasive transverse aortic constriction (MTAC) is a more desirable method for the constriction of the transverse aorta in mice than standard ...

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

微创肌肉植入 (MIME)--一种促进供体细胞介导的肌的新实验技术

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

小鼠横主动脉狭窄诱导左心室肥厚的技术研究

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

小鼠闭合性股骨骨折的形成: 骨愈合模型的研究

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

电磁导航经胸结节定位用于微创胸外科手术

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...