这项研究得到了国家研究基金会 (NRF-2014R1A2A1A11052042; 2015M3A9B4067068)、大韩民国科学技术部、韩国健康技术研发 & 开发项目 (HI16C1012)、卫生部 & 的资助。福利, 大韩民国, 和世大学医学院的 "Dongwha" 教职员研究援助计划 (6-2016-0126)。

所有的动物都被安置在一个标准的笼子里 (27 x 22.5 x 14 厘米3) 在实验室动物保育协会 (AAALAC) 的认证和给予食物和水ad 随意在交替 12-h 光/暗周期.作者遵循动物保护条例, 实验程序由世大学医学院 (IACUC No. 2010-0252; 2013-0220) 机构动物保育和使用委员会批准。
1. 小鼠新生儿脑损伤模型
<ol><li>用异氟醚麻醉幼崽。<ol>
<li>将幼崽 (少于 5) 放入麻醉盒中, 并关上盖子。</li>
		<li>打开麻醉系统约15分钟;用台式麻醉机调节气体和异氟醚。调整氧气流量计为1.5 升/分, 调整的异氟醚蒸发器到3-5% 的诱导麻醉。</li>
		<li>15分钟后, 将异氟醚蒸发器调整到 1-2%, 用于麻醉的维护。</li>
	</ol>
</li>
	<li>在解剖显微镜下放置一个完全麻醉的幼崽 (面向研究员的腹部) 并用胶带固定。</li>
	<li>用消毒剪刀在颈部做一个0.7 毫米的切口。</li>
	<li>用灭菌钳将脂肪组织小心取出, 并暴露一侧右颈动脉。</li>
	<li>结扎5-0 可吸收缝合的单侧右颈动脉。</li>
	<li>用5-0 缝线缝合颈部的切口。</li>
	<li>将每只幼崽放在一个37° c 温暖的缺氧室中, 用于1小时的恢复。请勿关闭室盖。</li>
	<li>手术后1小时, 当幼崽完全清醒时, 关闭缺氧室盖, 降低气体水平以建立缺氧状态 (8% O2和 92% N2)。</li>
	<li>90分钟的缺氧后, 将幼崽返回笼子。</li>
	<li>脑损伤后一周, 重复步骤1。<ol>
<li>麻醉后, 用无菌剪刀和镊子在头皮上做一个切口, 以确定右半球后外侧的脑损伤。 注意: 这种疗法能诱导幼崽缺氧。所有小鼠脑损伤的存在和程度, 通过肉眼观察, 通过半透明的头骨进行目测评估。根据皮肤变色的大小或体积 (即脑部病变), 幼崽被分为组。如果没有明显的皮质损伤, 则将小鼠分为 "无皮质损伤" 组。如果有明显的皮质损伤 (即右半球的病灶), 则将小鼠分为 "皮质损伤" 组。由于对小鼠的分类是在手术后一周完成的, 所以当大脑样本的形态在牺牲时1、2、3中明确定义时, 可以对分组进行修改. 4。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55838/55838fig1v3.jpg"> 图 1: 对小鼠脑损伤进行建模。 (a) 一只七天大的小鼠接受了手术, 一侧右颈动脉结扎。(B) 幼崽被放在缺氧室中90分钟, 8% O2和 92% N2。(C、D 和 E)新生儿 HI 损伤的脑部表现出不同程度的损伤, 并根据损伤程度进行分类。在14周, 大脑得到了, 病变是可视化的。(C) 被归类为 "无皮质损伤" 的大脑的图像。(D) 和 (E) 分为 "皮质损伤" 组。(F、G 和 H)分别对 (C)、(D) 和 (E) 小鼠进行有代表性的 MRI 检查。(F) 海马的损伤用黄色箭头表示, 右半球的病灶也用黄色箭头 (G 和 H) 表示。缩放栏 = 1 mm<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55838/55838fig1v3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
2. 新生儿行为测试
注意: 在这里, 行为测试是在6周的年龄进行的。
<ol><li>
被动回避任务。 注意: 为了评估基于学习和避免厌恶刺激的记忆功能, 两个隔间步进拍应进行13,14,15,16。<ol>
<li>将一只小鼠放入有机玻璃梭箱 (41.5 x 21 x 35 cm3) 的明亮车厢中。</li>
		<li>三十年代以后, 打开断头台门并且记录延迟时间为老鼠移动入黑暗的隔间 (由 300s)。</li>
		<li>关闭断头台门时, 所有的鼠标四四肢完全在黑暗的车厢内。</li>
		<li>管理电动脚休克 (0.5 毫安) 为2秒, 并将鼠标返回到它的笼子。</li>
		<li>在电动脚休克后, 在明亮的隔间中更换鼠标24小时。</li>
		<li>十年代把老鼠完全放在明亮的车厢里后打开断头台的大门, 并记录下鼠标进入黑暗车厢的等待时间 (高达 300s)。</li>
	</ol>
</li>
	<li>
阶梯行走试验。 注意: 阶梯梯级步行任务允许通过结合定性和定量分析熟练的步行17,18来区分运动功能的细微干扰。<ol>
<li>打开摄像机。</li>
		<li>将鼠标放在梯子的开始板上, 然后立即开始录制。</li>
		<li>录制视频, 重点放在鼠标的四肢。</li>
		<li>当鼠标触及梯子的最后一个面板时停止录制。重复来回旅行四次。</li>
		<li>分析视频记录, 并手动计算每个前肢的单张数, 如下所示:<ol>
<li>在计算机上以慢速 (0.1x) 播放视频记录, 并手动计算步骤。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
握杆强度试验。 注: 握力测试采用握力计, 包括推拉式应变计。<ol>
<li>将握杆强度装置固定在压克力板上。</li>
		<li>将鼠标放在亚克力面板上, 并按住它的尾部。<ol>
<li>移动握着尾巴的手, 这样鼠标就能到达并握住仪器的金属线。</li>
		</ol>
</li>
		<li>允许多次试验, 直到鼠标握住三角形的金属丝 (直径2毫米);该仪器自动在克中登记峰值力。 注: 使用三试验的平均峰值力进行分析19,20,21。</li>
	</ol>
</li>
</ol>

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作者没有竞争利益。

在这项研究中, 我们诱发 HI 脑损伤的新生儿 P7-10 CD-1 小鼠, 并确定了脑损害的相关认知和运动缺陷。在这个过程中, 闭塞的单侧右颈动脉是至关重要的。在这一步, 动脉可能被破坏和撕裂。大多数经历过动脉撕裂的幼崽都死了。反之, 如果研究人员结扎了另一条血管而不是单侧右颈动脉, 小狗的大脑只轻度受损, 没有明显的表型可观察到24。
在这项研究中, 由于小鼠和病变体积的变化, 大脑被分为几个组 (图 1 c-H)。一些轻度脑损伤的小鼠只在海马区受损, 而不损伤皮质区域 (图 1F)13。相反, 一些严重受损大脑的老鼠失去了大部分右半球, 皮质严重受损 (图1G 和 H)。因此, 研究人员应在程序19,25之后的一周内确定病灶的大小。由于使用 MRI 扫描对大脑进行评估, 因此对病灶体积和大小的测定更加可靠。因此, 我们建议研究人员用 MRI 来评价大脑, 尽管肉眼检查也是可行的。
脑瘫通常发生在幼儿期, 每1000名儿童的发病率约为两个病人5。由于新生儿 HI 小鼠模型可能是脑瘫或新生儿中风的典型模型4,11,26, 本研究的基线信息可用于脑瘫的临床前研究或新生儿中风
神经行为评估有助于识别认知和运动缺陷的表型13。本研究中引入的神经行为评估也适用于其他神经退行性疾病, 如亨廷顿氏症、帕金森病等。研究人员应该意识到, 在 PAT 期间, 受试者会受到电击。因此, PAT 应该最后执行, 所以电击不会影响其他行为评估。
为了进一步研究, 研究人员需要研究一个假手术组与 HI 组比较。对于一个特定的对照组, 研究人员可以在颈部进行切口, 并在没有任何动脉结扎的情况下闭合切口。为了模仿 hi 的操作, 这些幼崽应该放进缺氧室, 但没有缺氧, 在回到笼子之前, 它们的时间和 hi 组一样多。

新生儿 HI 脑损伤发生在幼儿期 (约两个病人每1000儿童)1,2,3,4,5。关于新生儿 hi 脑损伤的研究是重要的, 利用建立的新生儿脑损伤小鼠模型可以促进脑损伤的临床前期研究. 
传统的 HI 模型用于成年大鼠6。对于新生儿模型, Vannucci 法是常用的 P7 大鼠7,8。然而, 因为老鼠和老鼠是略有不同的9,10, 即使它们都是啮齿类动物, 我们在 P7-10 上对 CD-1 幼崽进行了改良的大米 Vannucci 方法, 根据先前的研究表明 P7-10 是未成熟的突, 对应于人类术语 P011,12。通过单侧颈动脉结扎和小鼠对 P7-10 幼犬缺氧8% 氧的暴露, 建立新生儿 HI 小鼠模型。
受手术影响的小鼠在右半球的后外侧区域显示出不同程度的脑损伤。为了识别认知和运动缺陷, 进行了基于 PAT、阶梯行走试验和握力测试的神经行为评估。分析了手术 (即正常) 和 HI 小鼠之间的差异。这项工作提供了关于 HI 鼠标模型的基本信息。MRI 图像显示不同的表型, 根据脑损害的严重性, 使用运动和认知测试分开。

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			<td>Jeung Do Bio &#38; Plant Co</td>
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			<td height="25" style="height:25px;">The ladder rung walking&#160;</td>
			<td>Jeung Do Bio &#38; Plant Co</td>
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			<td height="25" style="height:25px;">SDI Grip Strength System&#160;</td>
			<td colspan="2">San Diego Instruments Inc.</td>
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			<td height="25" style="height:25px;width:363px;">Grip-Strength Meter</td>
			<td>Ugo Basile&#160;</td>
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			<td height="25" style="height:25px;">Harvard Apparatus Fluovac anesthetizing system&#160;</td>
			<td>Harvard Apparatus</td>
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			<td>Orient Co., Ltd.</td>
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			<td height="25" style="height:25px;">Blue Nylon Mono Non-Absorbbable suture 5-0 50cm</td>
			<td>Ailee Co., Ltd.</td>
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			<td>IBM</td>
			<td>Ver. 23</td>
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neurobehavioral assessments in a mouse model of neonatal hypoxic-ischemic brain injury

我们对 CD-1 小鼠进行了单侧颈动脉闭塞, 以建立新生儿缺氧缺血 (hi) 模型, 并通过研究这些小鼠的神经行为功能与手术 (即:正常) 小鼠。在这项研究中, Vannucci 的方法被用来诱导新生儿脑损伤在产后7-10 天 (P7-10) 小鼠。通过单侧颈动脉结扎和暴露于缺氧 (8% O2和 92% N2为 90 min) 对幼犬进行 HI 操作。手术后一周, 受损的大脑用肉眼通过半透明的头骨进行评估, 并根据缺席 ("无皮质损伤" 组) 或皮质损伤的存在 ("皮质损伤" 组) 进行分组。比如右半球的病灶在6周, 进行了以下神经行为测试, 以评估认知和运动功能: 被动回避任务 (PAT), 阶梯行走试验和握力测试。这些行为测试有助于确定新生儿 HI 脑损伤的影响, 并用于其他神经退行性疾病的小鼠模型。在这项研究中, 新生的 HI 脑损伤小鼠显示出与右半球损伤相对应的运动缺陷。行为测试结果与人类新生儿脑瘫或新生儿中风患者出现的缺陷有关。在这项研究中, 建立了一个小鼠脑损伤模型, 并显示了不同程度的运动缺陷和认知障碍相比, 手术的老鼠。这项工作提供了关于 HI 鼠标模型的基本信息。MRI 图像显示不同的表型, 根据严重的大脑损伤的运动和认知测试分开。

所有数据都被表示为平均±标准误差 (SEM)。通过对 SPSS 统计软件进行独立或配对的t测试, 对两个组之间的变量进行比较。p值 < 0.05 被认为具有统计学意义。
新生儿 HI 损伤的大脑表现出不同程度的损伤, 并据此分类 (图 1C-E)。大脑是在14周获得的, 病变是可视化的。图 1C显示一个大脑被归类为 "无皮质损伤" 的大脑,图 1D显示一个被归类为轻度损伤的大脑,图 1E显示严重受损的大脑。(D) 轻度和 (E) 严重损伤分为 "皮质损伤" 组。在 HI 手术后, 13 周大的小鼠使用 MRI 成像, 结果 (图 1F-H) 分别是 (C)、(D) 和 (E) 损伤的典型图像。尽管在大脑的形态学上没有明显的损伤, 但 MRI 图像显示海马损伤 (图 1F)。海马体损伤 (图 1F, 用黄色箭头表示) 在轻度损伤的大脑中略显明显。在严重受损的大脑中, 鼠标失去了大部分右半球 (图 1G和 H, 用黄色箭头表示)。
由于高损伤的大脑显示海马损伤 (图 1FH), 与正常小鼠相比, hi 损伤的小鼠表现出记忆力减退。PAT 性能与海马损伤密切相关13,15,16,19。图 2显示, HI 损伤的小鼠比正常的小鼠有更多的认知缺陷13(正常 n = 10;你好 n = 9)。在正常小鼠的基线和24小时记忆测试之间有统计学意义的差异, 如图 2A (*p = 0.003 基于配对的t测试) 所示。图 2B显示了与正常小鼠相比, 高伤害小鼠认知功能的变化 (δ) 是基线和 24-h 测试之间的差异)13。
由于只有右半球受损, 新生儿 HI 脑损伤小鼠表现为偏瘫运动功能。相对于每个前肢所采取的步骤的总数, 阶梯横阶上的滑动百分比的差异是用来比较正常小鼠与新生儿 HI 脑损伤小鼠17,19。图 3显示高脑损伤小鼠对侧前肢的滑移率明显高于正常小鼠 (正常 n = 19;喜 n = 18;*p = 0.010 基于独立的t测试)22, 但在同侧前肢(p = 0.798 基于独立的t测试) 中没有观察到差异。
此外, 由于握力涉及大脑中的运动皮层, 正常和皮质损伤组显示握力的差异。虽然握力试验结果显示正常和无皮质损伤小鼠 (图 4A; 正常的 n = 4; 没有皮质损伤 n = 12), 图显示对侧前肢的握力显著皮质损伤小鼠比正常小鼠弱 (图 4B; 正常 n = 4; 皮质损伤 n = 36; *p = 0.036 基于独立的t测试)21,22,23。

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55838/55838fig2v2.jpg"> 图 2: 轻拍新生儿 HI 脑损伤和正常小鼠。 (A) 在明亮的车厢内测量和比较新生儿 HI 脑损伤与正常小鼠之间的潜伏期时间 (n = 9 和 n = 10, 分别)。(B) 电子电击时的测量被认为是基线, 在电击后24小时内对长期记忆进行了评估。Delta (Δ) 24 h 滞后时间是在 24 h 和基线下计算的函数之间的差异。*p< 0.05;所有数据均以平均± SEM 表示. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55838/55838fig2large.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/55838/55838fig3v2.jpg"> 图 3: 阶梯行走试验中的前肢滑移率。 对正常和 HI 脑损伤小鼠的对侧和同侧前肢滑移率进行了评价 (n = 19 和 n = 18, 分别)。*p< 0.05;所有数据均以平均± SEM 表示.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55838/55838fig3large.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/55838/55838fig4v2.jpg"> 图 4: 新生儿 HI 脑损伤和正常小鼠握力试验。 对侧前肢的握力进行了评估, 并比较了(A)正常, 无皮质损伤和 (B) 皮质损伤小鼠 (n = 4, n = 12, n = 36)。*p< 0.05;所有数据均以平均± SEM 表示.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55838/55838fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury at JoVE.com

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

我们进行了单侧颈动脉闭塞产后 7-10 CD-1 小鼠幼崽创建一个新生儿缺氧缺血 (hi) 模型和调查的影响, hi 脑损伤。我们研究了这些小鼠的神经行为功能与手术正常小鼠相比。

小鼠新生儿缺氧缺血性脑损伤模型的神经行为评估

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

neurobehavioral-assessments-mouse-model-neonatal-hypoxic-ischemic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.2K 次观看.  Yonsei University College of Medicine. 我们进行了单侧颈动脉闭塞产后 7-10 CD-1 小鼠幼崽创建一个新生儿缺氧缺血 (hi) 模型和调查的影响, hi 脑损伤。我们研究了这些小鼠的神经行为功能与手术正常小鼠相比。

视频: Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

侧向液压冲击:创伤性脑损伤小鼠模型

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

科研

神经科学

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

方法对敌人造成闭合性颅脑创伤性脑损伤<em&gt;果蝇</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

使用新颖的方法在自由移动的新生大鼠中记录脑电图

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

一种新的脑外伤体损伤模型

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

爆炸性神经损伤对压生啮鼠中脑动脉的影响

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for infants with the neurological sequela of prematurity. Ferrets are ideal candidates for modeling the preterm human brain, as they are born lissencephalic and develop gyrencephalic brains postnatally. At birth, ferret brain development is similar to a 13 week human fetus, with postnatal-day (P) 17 kits considered to be equivalent to an infant at 32&#8211;36 weeks&#39; gestation. We describe an injury model in the P17 ferret, where lipopolysaccharide administration is followed by bilateral cerebral ischemia, hypoxia, and hyperoxia. This simulates the complex interaction of prolonged inflammation, ischemia, hypoxia, and oxidative stress experienced in a number of neonates who develop brain injury. Injured animals display a range of gross injury severity, with morphological changes in the brain including narrowing of multiple cortical gyri and associated sulci. Injured animals also show slowed reflex development, slower and more variable speed of locomotion in an automated catwalk, and decreased exploration in an open field. This model provides a platform in which to test putative therapies for infants with neonatal encephalopathy associated with inflammation and HI, study mechanisms of injury that affect cortical development, and investigate pathways that provide resilience in unaffected animals.

The method describes inflammation-sensitized hypoxic-ischemic and hyperoxic brain injury in the P17 ferret to model the complex interaction between prolonged inflammation and oxidative brain injury experienced in a number of late preterm infants.

炎症敏感晚期缺氧缺血性脑损伤的Ferret模型

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

神经干细胞向大鼠和小鼠大脑的动脉内传递:对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...