We thank Katherine Clark for technical assistance. A.D.R.G. is funded in part by 5K01MH097957-03

成人（3-4个月）雄性小鼠的混合C57BL / 6背景是在这个协议中使用。动物保持在12小时光照/黑暗周期，并允许自由摄取食物和水。在此协议下的所有程序都按照批准的德雷克塞尔大学机构动物护理和使用委员会的协议进行的。 1.准备手术区<ol><li>消毒手术台上，用70％的乙醇，然后覆盖整个手术台上，吸收垫，并安排手术器械相邻立体。 </li><li>建立立体设备无机械手臂。安排上的立体定位的加热垫，并设置为37℃。避免通过将一小片纸巾或手术垫的动物和加热垫之间过热的动物。 </li><li>使用高压灭菌剪刀，切小块蒸压明胶海绵到含有0.9％无菌盐水的溶液，直到意图的无菌培养皿DY使用。 注：使用蒸压文书和无菌手术耗材保持无菌的工作​​环境。在过程或在通过浸渍到珠灭菌10-15秒，可以根据需要重新消毒的手术器械的动物之间。保持干净的手套在整个过程通过摩擦的手用70％的乙醇，根据需要，进行消毒。 </li></ol> 2.鼠标预学习外科手术<ol><li>从家笼中取出鼠标和重量（克）。 </li><li>鼠标放置到异氟醚诱导室，并设置氧气2升/分钟，异氟醚蒸发至5诱导麻醉手术平面，约3-5分钟。监测呼吸减慢和固定。检查鼠标使用的是脚趾捏反射完全镇静。 </li><li>当鼠标被完全镇静，放置在立体框架，将鼻的鼻锥，其中附有油管的异氟醚。插入耳棒插入耳道，并拧紧，保证了头部是稳定的。 </li><li>从耳刮头部的耳朵，并且从眼睛之间到耳朵后面。 </li><li>消毒用异丙醇和优碘碘溶液，每次3倍的交替湿巾皮肤。 </li><li>应用人工泪液来双眼，以防止它们在外科手术过程中变干。 </li></ol> 3.手术过程<ol><li>按捏脚趾或尾部监测麻醉深度。鼠标是在合适的手术飞机的时候没有反应，呼吸缓慢而均匀。 </li><li>使矢状窦旁皮肤切口从刚才的眼神几乎用11号手术刀片在一个单一的，坚定的运动两耳之间的后面。移动肌肤一边夹右侧有止血。 </li><li>覆盖膜使用11号手术刀和棉花的暗面颅骨清除涂药。或者，擦拭头骨棉签蘸0.9％生理盐水。允许完全干燥。 </li><li>我们荷兰国际集团的小尺，标记开颅在1毫米尾椎冠状缝，和开颅在1毫米左边缘的前缘横向于矢状缝（ 图1），用一个永久性标记。然后标记开颅的右侧和尾部的边界处从矢状面和冠状缝线，分别（ 图1）4毫米。 </li><li>使用0.5毫米钻头，开始钻慢慢继永久性标记轮廓进行开颅手术。一定不要通过头骨打破完全。轻轻按压在隔离片顶骨与5号钳，薄弱环节将让位给的压力。当薄骨是整个周长足够弱，骨片是准备拆除。 注意：如果调查的经验困难除去骨在一块，这表明骨没有充分钻孔期间变薄。考虑在随后的动物facilita进一步钻探头骨忒容易去除的骨片。 </li><li>使用10毫升注射器配有23政针，将少量0.9％盐水浸泡分离骨和钻孔区域。 </li><li>附加操纵臂的立体定位设备。附加一个新的第11号解剖刀刀片的探针架座具有面向吻侧刀片的锋利边。 注意：虽然喙和尾部组织遇到刀片的锋利和钝边，分别引起的穿透性损伤的机械损伤是整个病变的程度相当。我们观察到反应神经胶质增生的主要特征包括上调GFAP表达或扩散，喙和尾部部分之间没有明显的差异。 </li><li>保持机械臂出的方式，小心抬起使用5/45角度的钳分离骨。插入镊子进入分离骨和升降机的一侧的前端，使用杠杆拉断的骨片在一个FUL留下升运动。 注意：要小心，不要刺伤大脑或干扰颅骨下方的硬脑膜。 </li><li>取一小块浸泡吸收凝胶泡沫，并放置在裸露的大脑，以防止其干燥和吸收血液中任何可能存在的。 </li><li>一旦凝胶泡沫就位，摆动操纵臂到位并调整刀片开颅中心在凝胶泡沫。取下凝胶泡沫，降低叶片直至尖端接触硬膜，而不刺穿硬脑膜。马克背/使用游标刻度上的立体的纵臂腹坐标。 </li><li>使用机械臂，慢慢放下刀精确3毫米进入大脑。这是通过使用游标刻度标记上的操纵臂实现。允许刀片留在原地5-10秒。移动立体手臂插脚喙移动到opposi前尾鳍三次使刀片到达开颅手术的喙和尾部的边界TE结束。 注：硬膜之前不插入刀片拆除。与此相反的大鼠硬脑膜，这是〜80微米厚7，鼠标硬脑膜是相当薄（只有几个细胞层厚度）和插入过程中不会产生对解剖刀片的明显的阻力。使用一个新的手术刀刀片每只小鼠以确保每个动物接受了一致的损伤。 </li><li>慢慢抬高手臂立体，去除脑部的刀片。去除叶片后，立即放在脑表面另一块明胶海绵来吸收多余的血液或体液。 </li><li>同时，除去立体手臂和处置的第11号手术刀刀片。一旦出血停止后，取出明胶海绵。 </li><li>通过缝合与非吸收缝线，如ethilon或普理灵皮肤闭合伤口。下列手术缝线应去掉9-10天。 </li><li>返回鼠标其家笼，并让鼠标在加热系统缓慢复苏摹垫和监测遇险的任何迹象。回收异氟烷诱导麻醉通常去除的异氟烷后发生在2-5分钟。不要让动物无人看管，直到它已经恢复胸骨recumbancy。 </li><li>辖0.5-1毫升乳酸林格氏液皮下的，以确保水合作用。 </li></ol> 4.手术后护理<ol><li>回到殖民地之前密切监测动物手术后，直到恢复从麻醉。 <ol><li>为了减轻疼痛和不适术后，管理0.05-0.1毫克/公斤丁丙诺啡腹腔注射立刻过程如下。 </li><li>动物观察2-3天手术后遇险严重迹象，如限制运动，缺乏疏导，或体重减轻。安乐死的动物展示任何痛苦的这些迹象，并从研究中删除。 </li></ol></li><li>为了检查受伤组织的病理学，由Stan动物安乐死准心内灌注。 <ol><li>简言之，麻醉动物用氯胺酮/赛拉嗪的过量，则心内灌注15-20毫升的0.9％的NaCl，或直到肝清除血液，随后60毫升4％多聚甲醛在实验结束时。 </li></ol></li><li>转移到30％的蔗糖溶液之前，解剖的大脑和定位后2-4小时在4％多聚甲醛。或如加西亚8,9-描述部分的大脑上低温恒温器在40-60微米，并处理通过标准组织学或免疫组织化学程序。 </li></ol>

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The authors have nothing to disclose.

该颅骨或底层硬脑膜的钻井过程中不被损坏，这一点至关重要。使用轻的压力，同时钻探，以保证头骨不刺破。另外，应注意同时抬起头骨片，以确保硬脑膜不解除了与骨。 这里的前脑锐器伤描述模型穿透伤到中枢神经系统。虽然比TBI模型如FPI或CCI少临床翻译，前脑病变刺模型充当了广泛的研究，旨在通过调查离散的中枢神经系统损伤引发的各种生化，细胞，分子或事件的?...

A major challenge for developing successful therapies to treat the injured CNS is an incomplete understanding of the complex multicellular events that are triggered by the trauma. Reactive astrocytes are gaining increasing recognition as a promising target for novel therapies1. Though historically regarded as hostile to neural repair, reactive astrocytes are now recognized as critical components of a complex, multicellular neuroprotective response that includes attenuation of inflammatory processes and limiting secondary damage and neurodegeneration2-6. Although the neuropathological characteristics of reactive gliosis have long been well defined, the cellular and molecular mechanisms regulating reactive gliosis, and the diverse array of downstream consequences remain poorly understood. Understanding the mechanisms that drive reactive gliosis, as well as the subsequent cellular and molecular events, is an important step towards developing strategies aimed at promoting the neuroprotective properties of reactive gliosis, while attenuating the detrimental effects. 
Here we demonstrate a method to induce severe reactive astrogliosis in the forebrain of adult mice using a stab injury. In contrast to other traumatic brain injury (TBI) models, such as controlled cortical impact (CCI) or fluid percussion injury (FPI), which require specialized equipment to produce an injury, the forebrain stab requires only a stereotaxic device to stabilize the head and a No. 11 scalpel blade. Thus the forebrain stab lesion model is more broadly accessible to a wide range of laboratories that do not have access to the specialized devices necessary for creating an FPI or CCI injury. The method described here enables investigators to reliably and reproducibly trigger a robust gliosis response to investigate subsequent cellular and molecular events. Once recovered from surgery, animals that have received a forebrain stab injury can survive for prolonged periods without the need for specialized care and can be returned to the colony for acute, intermediate, or chronic studies. Though less clinically translatable than FPI or CCI models of TBI, a forebrain lesion produced by a stab injury serves as a simple yet useful experimental model to investigate basic biological mechanisms underlying reactive gliosis and other neuropathological events following trauma to the CNS.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Stereotax</td>
			<td>Harvard Apparatus</td>
			<td>726049</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed micro drill</td>
			<td>Harvard Apparatus</td>
			<td>724950</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel scalpel blade, #11</td>
			<td>MedVet</td>
			<td>JOR581S</td>
			<td></td>
		</tr>
		<tr>
			<td>5/45 angled forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam sponge 12cmx7mm</td>
			<td>Fisher</td>
			<td>NC9841478</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies and other reagents</td>
			<td>Manufacturer</td>
			<td>Catalog Number</td>
			<td>Dilution (brightfield) 
			Dilution (fluorescence)</td>
		</tr>
		<tr>
			<td>Rb anti-GFAP</td>
			<td>DAKO</td>
			<td>&#160;Z033429-2</td>
			<td>1:20k 
			1:1k</td>
		</tr>
		<tr>
			<td>Shp anti-BrdU</td>
			<td>Abcam</td>
			<td>ab1893</td>
			<td>1:2k 
			1:500</td>
		</tr>
		<tr>
			<td>Biotinylated goat anti-rabbit</td>
			<td>Vector Laboratories</td>
			<td>BA-1000&#160;</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Biotinylated rabbit anti-sheep</td>
			<td>Vector Laboratories</td>
			<td>BA-6000</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Alexafluor 488 goat anti-rabbit</td>
			<td>Life Technologies</td>
			<td>A-11008</td>
			<td>1:400</td>
		</tr>
		<tr>
			<td>Alexafluor 568 donkey anti-sheep</td>
			<td>Life Technologies</td>
			<td>A-21099</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>DAPI Nucleic Acid Stain</td>
			<td>Life Technologies</td>
			<td>D3571</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Cresyl Violet Acetate</td>
			<td>Sigma Aldrich</td>
			<td>C5042-10G</td>
			<td>1%</td>
		</tr>
	</tbody>
</table>

triggering reactive gliosis in vivo by a forebrain stab injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

由于动物接受该方法不需要专门的术后护理，短期或长期的时间生存期很容易被纳入研究，根据不同的需要进行调查急性或慢性的病理损伤后。反应性神经胶质增生的主要功能，如GFAP和体细胞的肥大的上调，可以早在2-3天损伤后观察到的。增生的星形胶质细胞反应的高峰期是在受伤后10天3-5。如下所示的代表性的结果是从该接收一刀病变7天早期动物。 前脑的以下一?...

Watch this Scientific Journal Video about Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury at JoVE.com

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

触发反应胶质细胞增生<em&gt;在体内</em&gt;由前脑锐器伤

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

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Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

10.4K 次观看.  Drexel University. This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods. 