作者感谢 Drs. 珍妮弗. 杨和亚历山大 (萨沙) 考夫曼为技术协助与这项技术并且许多有用和关键的讨论。血清激素测定是由2017在弗吉尼亚大学的生殖配体检测和分析核心研究中心进行的, 由尤尼斯肯尼迪发育研究院/NIH (NCTRI) 赠款 P50-HD28934 支持。
研究支持来源: R01 发育研究院 86100 (九巴), 成果管理制得到 T32 发育研究院007203的支持。

这里描述的方法是与国立卫生动物护理和使用指南的协议, 并已授权的机构动物护理和使用委员会在加州大学圣地亚哥分校。
1. 处理的驯化
<ol><li>处理过程<ol>
<li>把老鼠笼放在安全柜里。将第一只老鼠从笼子里移开, 放在安全柜内合适的表面 (如干净的笼盖上)。</li>
		<li>用一只手的食指和拇指握住尾部的底座, 轻轻地抑制工作表面上的鼠标。然后, 将尾巴的腹面, 从底座到尾端, 用食指和拇指牢牢地描出尾部。重复此抚摸6次 (即1 集), 并将鼠标返回到其笼中。</li>
		<li>等待6分钟并重复步骤1.1.2。 注: 总计, 步骤1.1.2 将重复4次, 每只鼠标 (即4套6笔画), 使每一个鼠标将收到总共24尾中风的每一天的处理。</li>
	</ol>
</li>
	<li>日常处理频率<ol>
<li>从五至计划采血日前两个星期, 处理鼠标, 如步骤 1.1, 每周五天 (小鼠在星期六或星期日没有处理, 直到最后两周的驯化)。</li>
		<li>如果在取样期间进行的实验性治疗将包括任何额外的处理 (如scruffing 或腹腔注射), 在每次处理过程中使老鼠适应这种处理活动。 注: 为适应注射程序, 使用钝针进行假注射。</li>
		<li>在计划采血日的两周内, 每周处理1.1、七天的老鼠。</li>
		<li>至少在计划的采血日前两周, 小鼠在配对时尽量减少对小鼠在取样过程中的干扰。</li>
	</ol>
</li>
</ol>2、采血管的制备
<ol><li>在玻璃瓶中制备超灵敏 LH 检测缓冲器: 0.2% 牛血清白蛋白和 0.05% Tween-20 在 PBS [0.2 克氯化钾, 8 克氯化钠, 1.44 克 Na2HPO4 (无水), 0.24 克的 PO2, 超纯水到4升, pH 1, 过滤器和蒸压釜], 贮藏在4°c。1 注: 超灵敏 LH 检测缓冲器的体积可根据样品数量和样品体积计算, 如下所示 (2.2 & 讨论)</li>
	<li>准备采血管 (0.6mL 离心管)。标签管和整除检测缓冲器 (每管57µL 缓冲器; 注意, 这一体积可能因所收集的血液量或血液与所需的缓冲液的比例而异)。管可以提前几天准备取样, 贮存在4°c。</li>
</ol>3. 收集血液
<ol><li>收集采血材料的准备工作。<ol>
<li>准备使用的生物安全柜, 并安排以下材料在敞篷或在附近的手推车:10 µL 吸管 (设置为3µL 或需要的血液收集量), 吸管提示, 垃圾桶的使用吸管提示, 定时器, 打印与动物数量和样本数 (有关取样或动物问题的说明), 带有采血管、剪刀和小纱布的冰桶 (2 "x 2")。 注意: 在实验室计时器上的 "计数" 功能是有用的, 因为它没有一个可以干扰老鼠的声音警报。</li>
		<li>使用永久性标记标记每个鼠标的尾部 (靠近底座), 以帮助在取样过程中快速识别正确的鼠标。</li>
	</ol>
</li>
	<li>尾部和采血前准备的修剪。<ol>
<li>在血液收集开始前45分钟, 将第一只老鼠从笼子里移开, 在安全柜内的适当表面 (干净的笼盖工作良好)。</li>
		<li>使用锋利, 无菌剪刀删除1-2 毫米的鼠标尾部的尖端, 以下无菌制剂 (即用酒精和 betadine 擦洗)。 注: 这可以在清醒的动物, 温和克制或在异氟醚麻醉下完成。 此外, 还可以根据当地 IACUC 的建议提供镇痛。</li>
		<li>小心地抚摸鼠标的尾部如上所述 (1.1.2; 1-2 冲程通常是足够的), 直到血滴在尾部尖端形成。</li>
		<li>使用一个干净的提示吸管收集3µL 的血液从尾部尖端和丢弃的吸管尖端与血液。</li>
		<li>在把老鼠送回笼子之前, 要确保血流停止。如有必要, 用无菌纱布轻轻按压。</li>
		<li>对所有老鼠重复步骤 3.2.1 3.2.5。随后的血液样本可以通过牢牢地抚摸老鼠的尾巴来收集, 直到尾巴尖端形成一滴血。 注: 如果血滴在结实的笔触后不形成, 尾巴可以用盐水浸泡的纱布垫擦拭。血栓更可能形成在不太频繁的取样 (即 > 15 分钟间隔)。一旦血栓从静脉中清除, 取样就可以继续进行。</li>
		<li>继续收集每只老鼠3µL 的血液, 如上文所述, 每6分钟约45分钟, 在这个前采血准备期间。扔掉血和吸管尖。</li>
	</ol>
</li>
	<li>采血<ol>
<li>从尾端收集3µL 的血, 如上文所述。注意确保将整个3µL 样品带入吸管尖端 (没有气泡或床上用品污染)。将血液样本喷射到标记管中的化验缓冲器中。混合通过反转, 并返回到冰的管。</li>
		<li>在把老鼠送回笼子之前, 要确保血流停止。如有必要, 用无菌纱布轻轻按压。</li>
		<li>对每个鼠标重复步骤 3.3.1 3.3.2, 每隔6分钟为预定的取样持续时间。监测每一个动物的窘迫迹象 (例如, 部分闭合的眼睑, 驼背的位置 [不是由其他治疗引起的) 或缺乏对处理的反应) 和终止抽样, 如果必要的话。 注意: 行为困扰或其他需要终止取样的并发症在我们的实验室极少发生。</li>
		<li>如果动物在血液取样后不被安乐死, 请确保尾部的血流完全停止。将每只动物笼内的溅出的血液擦掉 (或更换笼子), 并将动物笼返回到外壳架上。</li>
	</ol>
</li>
</ol>4. 样品处理和分析
<ol><li>将血液样本储存在-20 摄氏度直到分析。 注: 在检测缓冲液稀释的全血标本中, LH 测定;在贮存或分析之前, 不需要分离血清或血浆。</li>
	<li>用超灵敏 ELISA 法测定血液样本, 如前所述1 , 并报告全血中 ng/毫升的值, 并对测定缓冲区中样品容积的稀释进行校正。</li>
	<li>识别数据集中的 LH 脉冲。根据数据集的不同, 计算和比较平均脉冲振幅和脉冲频率 (或脉间间隔)。 注: 已发布标准13,14和程序,15,16检测 LH 脉冲可用。</li>
</ol>

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作者没有什么可透露的。

在这里, 我们描述了一个经常性的血液收集的全血尾端样本, 以评估脉冲 LH 分泌的小鼠的协议。该协议可以收集样本, 以检测在受心理社会压力照射后搏动性 lh 分泌物的急性变化, 并且很适合在其他急性或慢性操作下对 lh 脉冲进行评估。
该协议的关键组成部分, 以实现可靠和健壮的 LH 的配置文件是处理驯化期。在文献中推测, 处理压力会损害激素分泌的想法在17</...

哺乳动物性腺功能依赖于促性腺激素分泌, 促黄体激素 (LH) 和卵泡刺激激素, 从垂体腺。促性腺激素是以脉动或浪涌模式分泌促性腺激素释放荷尔蒙 (促性腺素) 的下丘脑分泌物。LH 和 FSH 的合成和分泌都是通过内分泌、分泌和分泌作用来调节的, 包括下丘脑促性腺激素、性腺激素和激活-抑制素-卵泡抑素系统, 以及无数包括应力和能量平衡在内的生理条件。2
lh 在血液中的脉动模式产生于一个相当突然的 lh 放电到外周血, 其次是近似指数消除。该模式的重要特征包括每个 lh 放电的频率和 lh 反应的振幅, 这两者都是由释放促性腺激素. 由于收集下丘脑-垂体门血的困难, 测量搏动性促性腺激素, LH 的取样和测量被用来作为促性腺激素调节下丘脑-垂体-性腺轴的代表。因此, 关键信息编码的频率和幅度的 LH 脉冲, 这不能确定从一个单一的样本。
对搏动性 LH 分泌物的分析历史上仅限于大型哺乳动物 (人类、灵长类动物和绵羊), 原因是它们的血容量大, 而且对频繁的血样采集有耐受性。在啮齿动物中, 频繁的血液取样仅限于大鼠, 通过留置心房导管实现。3,4相对较低的遗传成本和可用性 (例如, CRISPR) 和复杂的神经回路 (如光遗传学、chemogenetics) 操作使小鼠成为具有吸引力的模型有机体;然而, 获得频繁的血液样本和随后的 LH 浓度分析, 直到最近, 证明是难以捉摸的。这项艰巨的任务是由 Steyn 和同事开创的。1从那以后, 几个实验室已经开始使用频繁的血液取样和超敏 lh 检测, 以评估脉冲 LH 分泌物在各种实验范式。5,6,7,8,9应指出的是, 寻求一种实用的方法来收集来自小鼠的多份血样已经进行了至少40年的10 , 并进行了多次改良。11,12
对 LH 脉冲模式 (即频率和振幅) 的评估, 是监测这种遗传听话动物模型中的基础促性腺激素分泌的一个重要的改进。传统上, 小鼠的 LH 浓度在单个血液样本中测定。单点样本的一个缺点是高度可变的数据集, 因为 LH 浓度在每个脉冲期间自然波动。另一个缺点是, 孤立的测量本身就忽略了 LH 脉冲分泌模式所传达的关键信息。因此, 在自由行为, 无约束的小鼠收集频繁的血液样本 (除了在取样过程中温和处理) 的方法将提供增强的信息, 并证明有用的许多实验室研究搏动激素调节。
在这里, 我们描述了一个协议, 收集频繁 (每6分钟) 血液样本从清醒, 无拘无束的老鼠。重要的是, 我们包括一个处理习得协议, 允许强健和连续检测脉冲 LH 分泌物在整个血液样本收集的时间内, 对急性挑战的前期和事后评估可以确定,如对固定化和克制的社会心理压力的反应。对全血标本 LH 浓度进行了有效的测定;1本协议的重点是收集血液样本的 LH 脉冲测量方法。

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			<td height="21" style="height:21px;width:216px;">Biosaftey cabinet</td>
			<td style="width:121px;">Lab Products Inc</td>
			<td style="width:119px;">L/F-B</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">A5403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">P2287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">P9333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4 (anhydrous)</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">S7907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">P5655</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrapure water</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:119px;"></td>
			<td>Purified and filtered water</td>
		</tr>
		<tr height="92">
			<td height="92" style="height:92px;width:216px;">Broome Rodent Restraint Device</td>
			<td style="width:121px;">Harvard Apparatus</td>
			<td style="width:119px;">52-0460</td>
			<td style="width:183px;">Not necessary for blood collection, but were used in the collection of representative data.</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">DynPeak</td>
			<td style="width:121px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:183px;">http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0039001</td>
		</tr>
	</tbody>
</table>

frequent tail-tip blood sampling in mice for the assessment of pulsatile luteinizing hormone secretion

在许多内分泌系统中, 循环因子或荷尔蒙不会连续释放, 而是作为一种离散的脉搏分泌, 以响应释放因子。单点取样措施不足以充分理解正常生理条件下或失调条件下脉动激素分泌模式的生物学意义。促黄体激素 (LH) 是由垂体前 gonadotrope 细胞合成的, 在脉动模式下分泌, 需要经常采集血液样本进行脉搏评估。这是不可能的, 直到最近, 由于开发了高灵敏度 LH 检测和先进的技术, 频繁的低量样本收集, 最初描述的 Steyn 和同事。1在这里, 我们描述了一个协议, 从小鼠频繁的外周血标本收集, 充分的处理驯化, 以检测 LH 的脉动分泌。目前的协议详述了一个扩大的驯化期, 允许评估的健壮和连续脉冲的 LH 在多个小时。在这个协议中, 尾巴的尖端被修剪, 血液是从尾部收集使用手持吸管。为了评估 gonadectomized 小鼠的搏动性 LH, 每隔5-6 分钟收集一系列样品, 90-180 分钟. 重要的是, 在清醒、自由行为的小鼠体内, 采集血液和测定 lh 的健壮脉冲, 给予适当的处理驯化和努力, 以尽量减少环境压力。在采血前4-5 周内可以达到足够的驯化。本议定书强调了方法的进展, 以确保收集全血样本, 以评估在老鼠的多小时内脉动 LH 分泌模式, 一个强大的动物神经内分泌研究模型。

典型的 LH 脉冲模式从4小鼠显示在图 1 (数据重印从杨等, 2017)。LH 是在频繁的血液样本测量, 以确定对急性社会压力挑战的反应。成年雌性 C57/Bl6 小鼠被卵巢切除, 并在本议定书中详细处理, 以便频繁采血。为所有动物的前90分钟收集血液样本, 以建立 LH 搏动分泌物的预处理基线。在取样的第二个90分钟内, 控制 (非受压) 动物留在笼子里, 限制压力...

Watch this Scientific Journal Video about Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion at JoVE.com

Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion

在这里, 我们提出了一个尾巴尖端血液取样协议, 以频繁样本收集在无约束的小鼠。该方法对评估脉动性促黄体激素分泌模式有一定的参考价值, 可用于其他循环因素的分析。

小鼠经常尾端采血对脉动性促黄体激素分泌的评价

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing ...

frequent-tail-tip-blood-sampling-mice-for-assessment-pulsatile

Research

JoVE Journal

Neuroscience

14.6K 次观看.  University of California, San Diego School of Medicine. 在这里, 我们提出了一个尾巴尖端血液取样协议, 以频繁样本收集在无约束的小鼠。该方法对评估脉动性促黄体激素分泌模式有一定的参考价值, 可用于其他循环因素的分析。

视频: Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the beam.

在小鼠体内的汽车平衡和协调的评估使用的平衡木

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

科研

神经科学

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

FITC 标记白蛋白静脉滴注对神经退行性疾病模型血脑屏障通透性的评价

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

一种体内评价大鼠缺血性中风模型中血脑屏障的破坏

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

间刺激间隔对啮齿类动物时间处理评估的力量

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

双直接注射血液到西斯特纳麦格作为苏巴拉奇内德出血的模型

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...