对此，我们深表感谢弗兰克博士基尔霍夫提供GFAP-EGFP和CX3CR1-EGFP小鼠品系。这项工作是由国际流动的中心芬兰，芬兰国家技术局，神经芬兰研究生院（FGSN）和芬兰科学院资助。

所有这里介绍的方法是按照动物保健（在动物实验二千○六分之六十二芬兰法）局部指引进行。动物许可证（ESAVI/2857/04.10.03/2012）由地方当局（ELÄINKOELAUTAKUNTA-ELLA）获得。 1-3月龄成年小鼠，体重24-38克，被关在单独的笼子中的认证大学的动物设施和提供食物和水随意 。 1。脑损伤成像通过颅窗口<ol><li>麻醉动物和准备操作字段<ol><li>通过腹腔注射氯胺酮（的Ketalar，80毫克/千克）和赛拉嗪（Rompun，10毫克/千克）溶解在过滤灭菌的磷酸盐缓冲盐水（PBS），pH为7.4的混合物麻醉小鼠。检查鼠标的反射规律（尾部和脚趾捏探头），以验证麻醉深度。增加剂量合适时，保持适当的麻醉，但要避免overdOSE。 </li><li>外科手术，成像会议和第一个小时从麻醉后恢复过程中使用加热垫维持动物在37.0°C。 </li><li>为了保护鼠标的眼睛干涩，敷眼润滑剂。 </li><li>施用地塞米松（Rapidexon兽医，2毫克/千克）经皮下注射，以减少炎症和脑水肿。 </li><li>之前或紧接在手术后给药的镇痛（例如，Ketoprophene腹膜内）达30分钟。如果动物出现任何疼痛症状，如不愿动，吃或喝，或体重下降，流涎，竖毛，或异常呼吸音手术后，重复注射止痛药。 </li><li>刮胡子鼠标的头部用剃齿机（见材料和​​设备）。避免损坏晶须。 </li><li>治疗对小鼠的头部皮肤用70％乙醇溶液擦拭剃区域。 </li><li>用手术剪（见材料和设备）和镊子（参见材料和设备），切断从耳朵之间的中间线的皮肤到前额。 </li><li>轻轻刮掉附着在头骨用钝刀刃显微结缔组织。 </li><li>皮肤滑动边界侧身拉耳棒略成骷髅的头部和皮肤固定耳窍。 </li><li>清洁用无菌PBS头骨和治疗用0.5％氯己定二葡糖酸盐手术部位对小鼠的头部，然后用无菌棉签和压缩空气的组合干燥颅骨表面。 </li></ol></li><li>挑刺的伤害加刑<ol><li>位置的小动物立体定位仪与动物持有人（见材料和​​设备）。 </li><li>调节双目显微镜的位置旁边的立体定位仪集中动物的颅骨的表面上。 </li><li>使用双目显微镜，定位在头骨的前囟门点。 </li><li>识别区域选择性n个采用立体坐标的兴趣。 注意：避免利益这些领域是直接在位于皮质​​浅表血管。这些破坏可以强烈地影响到创伤的进展。 </li><li>放置30 G针所选择的区域之上，并通过抓颅骨表面标记目标网站。 </li><li>钻颅骨与所有可能的预防措施，以避免受影响骨骼的表面积不必要的扩大。在显微镜下使用高速手术电钻（见材料和​​设备）。停止，如果液体出现在钻孔钻探区域。 </li><li>轻触钻探井的底部用针。 </li><li>顺利沾针入脑（入纬率5〜10毫米/分钟），以0.5-2.0毫米根据芒刺申请地点的坐标的深度。 </li><li>达到所需深度（回缩率5〜10毫米/分钟）后，立即取出针和擦拭血用小棉球扎后出现（SEË材料和设备）。 </li><li>用微量注射泵（WPI）和10微升汉密尔顿注射器组装与玻璃吸管执行100微米基罗丹明101显微注射或感兴趣的另一种化合物进入病灶部位。 注：在制备硼硅玻璃毛细管的玻璃移液管，锐化尖端的直径为10-20微米。 </li><li>注入250-1,500 NL注射溶液在2-5升/秒的速率。 注意：保持移液管输液结束后至少5分钟，脑实质，然后删除它慢慢地，轻轻地，防止液流出。 </li></ol></li><li>开颅手术治疗慢性颅窗<ol><li>钻骷髅轻轻地，小心地使周围损伤部位的圆形窗口。使用3-3.5毫米直径的窗口。 </li><li>申请一滴皮质缓冲液（125 mM氯化钠，5mM的氯化钾，10mM葡萄糖，10mM的HEPES，2mM的氯化钙和2mM MgSO 4干燥蒸馏水H 2 O）以覆盖无线网络程式视窗。 </li><li>对颅窗口位置的圆形玻璃盖玻片（＃1.5厚度）。 </li><li>除去周围的盖玻片多余的皮质缓冲。 </li><li>轴封采用聚丙烯胶盖玻片的边缘（见材料和​​设备）。 注：请确保胶水不会应用到盖玻片的上表面。如果玻璃盖被污染的胶水，等到玻璃稳定地粘在颅骨和在玻璃表面胶水变干，然后小心地用microblade去除污染胶水。 </li><li>等待5分钟，让胶水干起来。 </li><li>使用耳杆高度螺钉，调整头部位置，使得金属支架可以放置。 </li><li>钢支架的环（见材料及设备）上涂抹少量的聚丙烯胶。 </li><li>胶钢支架的环与金属支架手柄向后定向的玻璃盖玻片。 </li><li>等待5分钟，让胶水干起来。 </li><li> M九牙科水泥（见材料和​​设备）与聚丙烯酸胶在3.5公分培养皿（或模拟）来实现粘性条件。 </li><li>密封颅窗口的边界与水泥​​+胶混合物中，并覆盖具有相同的混合物到皮肤边界头骨的暴露表面上。 </li><li>等待15分钟，让水泥+胶水混合干起来，然后取下耳棒。 </li><li>进行双光子激发显微（TPEM）成像的感兴趣区域和控制区域，因为它在协议3所述。 </li><li>成像后，让动物从麻醉中恢复一个加热垫，并根据观察其保存在一个单独的笼子里，直到完全康复。湿的食物可以提供给便于咀嚼和水化。 </li></ol></li></ol> 2。脑损伤成像通过减薄骷髅<ol><li> Anaesthetizing动物和准备操作字段<ol><li>麻醉小鼠并准备创伤诱导如步骤1.1中所述。 </li></ol></li><li>颅骨变薄和挑刺的伤害施加某种<ol><li>位置的小动物立体定位仪与动物持有人（见材料和​​设备）。 </li><li>调节双目显微镜的位置旁边的立体定位仪专注于动物颅骨表面。 </li><li>使用双目显微镜定位前囟门处的颅骨，找出大脑区域基于立体定向坐标进行成像，并通过抓标记。 注：头骨变薄位置不应设在或邻近颅缝，如颅骨稳定性受到威胁在这些地区。此外，大型皮质或脑膜血管和位于下方的颅骨缝脑膜也容易造成成像伪影。 </li><li>将涂过胶水的对动物的头骨感兴趣的区域的金属支架，适用于轻的压力，并等待5分钟，直至金属支架牢固地粘在头骨。 </l我&gt; <li>混合牙科水泥（见材料和​​设备）与聚丙烯酸胶在3.5公分培养皿（或模拟）来实现粘性条件。 </li><li>密封该金属保持器的边界与水泥​​+胶混合物中，并覆盖具有相同的混合物到皮肤边界头骨的暴露表面上。 </li><li>等待15分钟，让水泥和胶水混合干起来。 </li><li>冲洗减薄头骨区域和所述金属支架的周围部分几次，用PBS，这样的非聚合胶的残余被洗去。 注：关键是要确保头骨稳定附着在持有人。这使得制剂稳定性成像过程中。 </li><li>用双目显微镜的高倍放大模式，取出颅骨的上层用高速微型钻头创建〜0.5-1.5毫米直径的薄壁头骨区域。钻去除骨碎片过程中，使用压缩空气。进行钻探intermittentl为了避免摩擦引起的过热减薄过程期间年。使用室温下的溶液冷却钻头并定期应用缓冲到减薄的区域吸收热量。 注意：为避免损坏皮质，不钻在大区域（&gt; 1.5毫米）下降到薄层（&lt;50微米）。 注：啮齿动物颅骨结构包括两薄层密质骨的由一层厚厚的松质骨的分离。海绵骨形态同心圆和小管，它含有血管的微小的孔穴。用微型钻头来删除这两个外部密质骨层和大部分的海绵层。的松质骨内的血管破裂可引起出血。使用胶原止血海绵制止这种出血。 </li><li>利用二次谐波产生（SHG）成像检查多数骨松质是否已被删除。要小心，避免过度变薄，确保剩下的骨头是个icker大于50μm这个阶段的准备。 </li><li>放一滴温暖缓冲液（35-37℃）对减薄区域的顶部。用显微叶片或Micro整理柏迪进一步去除骨层，直径为〜20微米厚的骨形成〜700微米的平滑区域。在此步骤中，它是有帮助的执行重复的SHG成像和定期测量的骨厚度。 </li><li>对感兴趣的区域和控制区域进行TPEM成像。 </li><li>使用耳杆高度螺钉在这样一种方式，减薄区域定位严格水平调整磁头位置。定位细化区域上方的针，并确保有针对性的网站下方没有大血管。 </li><li>通过浸渍在针入脑，以0.5-2.0毫米从减薄颅骨表面的深度，并根据刺应用部位的坐标使病变。 </li><li>取下针头，抑制出血后t出现他急性脑损伤与止血棉球（见材料和​​设备）。等到血液凝块的形式和血管搏动在损伤部位停止。 </li><li>执行100微米基罗丹明101或感兴趣的另一种化合物进入病灶部位注射，如第1.2.10如上所述。 </li><li>执行TPEM成像在协议3所述监视伤害的进展和恢复。 </li><li>成像后，让动物恢复从麻醉中意识上的一个加热垫。不要让动物无人值守，它只有经过充分的体力恢复返回到其家笼。 </li></ol></li></ol> 3。成像<ol><li>由金属件安装到定制的框架将动物的显微镜下。 </li><li>对于成像，使用如 FV1000MPE双光子显微镜配备埋汰DeepSee激光和XLPLN 25X 1.05 NA 体内双光子优化水浸泡的目的成像。 </li><li>确定病灶部位采用宽视场荧光模式在显微镜下。使用长通滤波器可视化脑血管，并根据血管的观察模式选择的控制区。 </li><li>到图像的二次谐波产生（SHG）信号，调谐飞秒激光的800nm​​的波长，并收集用380-410 nm的旁路过滤器所发射的光。对于荧光成像，使用带通滤波器（515-560纳米），以收集发射光，并且以下波长的光被用来激发荧光：GFP-860纳米，YFP-950纳米。 </li><li>使用Fluoview软件进行图像采集。 </li><li>存储坐标每投资回报率进行后续重复成像。图像相同的投资回报随着时间的推移，每次以最大限度地提高图像的重叠调整坐标。 </li><li>与适当的软件（ 例如 。ImageJ的）分析图像。是用带Imaris里的软件在这里提出重建。 </li></ol>

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作者什么都没有透露。

脑外伤是突然的，不可预知的事件。在这里，我们描述了再现的人类患者中观察到的脑损伤，如神经变性，消除枝晶，脑水肿，神经胶质疤痕，在大脑皮层再加焦蛛网膜下腔出血和出血的通透性增加后的病理变化的频谱的动物模型血 - 脑屏障。研究原发性和继发发病机制，以及创伤后恢复，此损伤模型，结合纵向细的神经元和神经胶质的结构体内的可视化。转基因小鼠系被用来表达?...

急性脑损伤与损伤的机动车事故高发一个显著的公共卫生问题，跌倒或袭击​​，以及随后的慢性残疾的患病率较高。治疗方法为脑损伤的治疗仍然完全对症，从而限制了院前急救，外科和重症监护的有效性。这使得脑损伤的社会和经济影响尤其严重。对于各种各样的原因，大多数临床试验中失败的使用新的治疗方法来证明改善脑损伤后的恢复。 动物模型是开发新的治疗策略对一个阶段，药物的功效可以在患者的脑损伤进行预测的关键。目前，头部外伤的几种公认的动物模型存在，包括控制皮层的影响1，液压冲击伤2，动态皮质变形3，体重下降4，和光损伤5。许多实验模型已被用于研究头部外伤相关病理的若干形态，分子和行为方面。但是，没有一个动物模型中验证新的治疗策略完全成功。脑损伤的可靠，重现性好，控制动物模型的发展是必要的，以评估复杂的病理过程。 最新显微成像技术和基因编码的荧光记者新颖的组合提供了一个前所未有的机会来研究脑损伤的各个阶段，包括原发性损伤，原发性损伤，继发性损伤和再生的蔓延。特别是， 在生物体内的双光子显微术是一种独特的非线性光学技术，允许细胞和甚至亚细胞结构中啮齿动物脑的深部皮质层的实时可视化。几种类型的细胞和奥尔加的nelles可以同时通过组合不同的荧光标记物进行成像。使用这个强大的工具，我们可以想像在创伤条件下活脑动形态和功能改变。在研究脑损伤体内双光子显微镜的优点由基洛夫和他的同事6最近被证实。使用温和的焦点皮质挫裂伤模型，这些作者发现，在pericontusional皮质树突急性损伤是由局部血流量下降门。此外，它们显示出，绕挫伤站点的代谢受损皮质进一步由扩频的去极化损坏。继发性损伤影响突触电路，使得创伤性脑损伤的后果更严重。 在这里，我们提出了立体刺的方法，用注射器针头，这可能与同时外用药物的应用相结合，为局部脑的先进模式损伤和作为研究急性创伤的病理生理学后果在哺乳动物大脑体内的工具。

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		<tr height="21">
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			<td style="width:119px;">M8410</td>
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			<td height="35" style="height:35px;width:216px;">Multiphoton Laser Scanning Microscope</td>
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			<td height="21" style="height:21px;width:216px;">polyacrylic glue</td>
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			<td style="width:119px;">Loctite 401</td>
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			<td height="52" style="height:52px;width:216px;">Small animal stereotaxic instrument</td>
			<td style="width:71px;">David Kopf Instruments</td>
			<td align="right" style="width:119px;">900</td>
			<td style="width:167px;"></td>
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			<td height="35" style="height:35px;width:216px;">Stoelting mouse and neonatal rat adaptor</td>
			<td style="width:71px;">Stoelting</td>
			<td align="right" style="width:119px;">51625</td>
			<td style="width:167px;">Assembled on stereotaxic instrument.</td>
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			<td height="35" style="height:35px;width:216px;">Student iris scissors, straight 11.5 cm</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">91460-11</td>
			<td style="width:167px;"></td>
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			<td height="35" style="height:35px;width:216px;">Sulforhodamine 101</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">S-359</td>
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			<td height="35" style="height:35px;width:216px;">UMP3 microsyringe pump and Micro 4 microsyringe pump controller</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">UMP3-1</td>
			<td style="width:167px;">Microinjector and controller</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Xylazine</td>
			<td style="width:71px;">Bayer Health Care</td>
			<td style="width:119px;">Rompun vet</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

acute brain trauma in mice followed by longitudinal two-photon imaging

虽然急性颅脑外伤往往是由于磁头损坏在不同的事故和影响了人口的很大一部分，目前还没有有效的治疗它。目前使用的动物模型的局限性阻碍了病理机制的认识。多光子显微镜可以学习完整的动物大脑在纵向生理和病理条件内的细胞和组织。在这里，我们描述了两种型号的急性脑损伤研究通过脑细胞行为的创伤条件下的双光子成像手段。一，选择脑区域受伤用锋利的针，以产生在脑实质受控的宽度和深度的损伤。我们的方法使用立体刺用注射器针头，它可与药物同时应用相结合。我们建议，这种方法可以作为一种先进的工具来研究细胞的急性创伤的病理生理学后果机制在哺乳动物大脑<em&gt;在体内。在这段视频中，我们结合急性脑损伤与两种制剂：颅窗口和颅骨变薄。我们还讨论了双方准备外伤后大脑再生的多区段成像的优点和局限性。

我们已经优化了两种操作步骤：1）慢性颅窗口和2）颅骨变薄，在转基因小鼠创伤后的脑成像。实验准备工作的示意图示于图1。外伤性刺由0.3 mm外径（30 G）的钢针施加到钻的井（ 图1A）。一个成功的颅窗口准备让成像深度可达650微米的软脑膜表面（ 图1B）以下，而颅骨变薄倾向于征收约300微米（ 图1C）的限制，这表现在Thy1系-YFP-的三维重建ħ小?...

Watch this Scientific Journal Video about Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging at JoVE.com

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

急性脑外伤是没有适当的治疗迄今严重的伤害。多光子显微镜允许纵向研究急性颅脑外伤的发展过程及探测的治疗策略，在啮齿类动物。两款车型的急性脑外伤的研究与体内双光子脑成像中演示了这个协议。

急性脑外伤小鼠其次是纵向双光子成像

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

acute-brain-trauma-in-mice-followed-by-longitudinal-two-photon-imaging

Research

JoVE Journal

Medicine

12.3K 次观看.  University of Helsinki. 急性脑外伤是没有适当的治疗迄今严重的伤害。多光子显微镜允许纵向研究急性颅脑外伤的发展过程及探测的治疗策略，在啮齿类动物。两款车型的急性脑外伤的研究与体内双光子脑成像中演示了这个协议。 

视频: Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Pseudofracture: An Acute Peripheral Tissue Trauma Model

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality.1-9 Many studies have begun to assess these changes in the reactivity of the immune system following trauma.10-15 
Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses.16-21
The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible.22-30
This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.
The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution.
Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. 31
Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. 32-36 The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. 37

Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.

Pseudofracture：急性周围组织的创伤模型

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; ...

科研

医学

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

挑战的药理功能磁共振成像：在临床前研究使用的5  - 羟色胺系统中的应用

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

超声成像引导下腔内注射到患乳腺癌脑转移其次是纵向的MRI的小鼠模型

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

纵<em&gt;在体内</em&gt;的Cerebrovasculature影像：相关性疾病CNS

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

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

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

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 ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Orthopedic Robot-Assisted Femoral Neck System in the Treatment of Femoral Neck Fracture

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm fluoroscopy to place the screw freehand and requires several guide wire adjustments, which increases the operation time and radiation exposure. Repeated drilling can also cause damage to the blood supply and bone quality of the femoral neck, which can be followed by complications such as screw loosening, nonunion, and femoral head necrosis. In order to make fixation more precise and reduce the incidence of complications, our team applied robot-assisted orthopedic surgery for screw placement using the femoral neck system to modify the traditional procedure. This protocol introduces how to import a patient's X-ray information into the system, how to perform screw path planning in software, and how the robotic arm assists in screw placement. Using this method, the surgeons can place the screw successfully the first time, improve the accuracy of the procedure, and avoid radiation exposure. The whole protocol includes the diagnosis of femoral neck fracture; the collection of intraoperative X-ray images; screw path planning in the software; precise placement of the screw under the assistance of the robotic arm by the surgeon; and verification of the implant placement.

This article introduces a method of robot-assisted orthopedic surgery for screw placement during the treatment of femoral neck fracture using the femoral neck system, which allows for more accurate screw placement, improved surgical efficiency, and fewer complications.

骨科机器人辅助股骨颈系统治疗股骨颈骨折

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm ...

Urethral Stricture Induction Followed by Buccal Mucosa Graft Urethroplasty in a Rat Model

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is necessary, although in some cases, it is inappropriate or needs to be optimized to repair a given stricture. Therefore, developing innovative procedures and evaluating their putative success in experimental models is crucial to fit the clinical need. With this goal, this study describes a protocol in which urethral stricture was induced by electrocautery in Wistar rats. Urethral reconstruction was performed 1 week later with a buccal mucosa graft, harvested from the lower lip, and placed in a ventral onlay fashion. A retrograde urethrogram showed a significant improvement in urethral diameter after urethroplasty compared to the respective value after stricture induction. Additionally, the graft placement was assessed by blood perfusion analysis using laser Doppler. As expected, a dark blue area corresponded to the non-vascularized buccal mucosa graft. This procedure can successfully simulate the normal pathophysiological process of urethral injury and tissue modulation, as well as urethral reconstruction using a buccal mucosa graft in a reproducible manner, and serve as the basis for future research based on tissue engineering or urethral grafts.

In the present protocol, a urethral stricture induction was developed in Wistar rats, followed by urethral reconstruction with a buccal mucosa graft. A retrograde urethrogram and laser Doppler assessment were performed, validating urethral reconstruction (after stricture formation) and graft placement.

大鼠模型中的尿道狭窄诱导后进行颊粘膜移植尿道成形术

Urethral reconstruction is an important area of expertise for urologists. The buccal mucosa is considered the best option when urethral grafting is ...