作者感谢阿尔达比勒医学大学 (阿尔达比勒, 伊朗) 研究的副校长为财政支持 (授予 No: 9607)。

所有的程序都按照阿尔达比勒大学医学研究委员会的指导方针进行动物研究 (道德 ID 号: IR。ARUMS。1394.08). 在这一可视化研究中, 我们使用了由牧场研究所 (伊朗德黑兰) 获得的成年雄性大大鼠 (300-350g)。
1. 麻醉和血流仪
<ol><li>麻醉使用4% 异氟醚, 并保持它与异氟醚 (1-1. 5%) 在一氧化氮 (70% 伏/五) 和氧 (30% v/v) 的混合物在手术期间。</li>
	<li>放置一个麻醉动物在一个反馈控制的加热毯的俯卧姿, 并保持体温在37±0.5 °c 通过一个直肠探头连接到这个加热单元。</li>
	<li>在两只眼睛上涂抹少量的眼膏, 以防止干燥。</li>
	<li>用电动快剪剃去头骨左侧的手术区。使用 betadine 溶液与纱布垫消毒皮肤。用含有70% 乙醇的无菌垫冲洗这个区域, 并重复这两个步骤三循环17。</li>
	<li>为镇痛, 注射0.2 毫升0.5% 布比卡因皮下入手术区域17。</li>
	<li>在头骨上做一个1厘米长的皮肤切口 (从左眼的侧眼角延伸到左耳的底部), 解剖左颞的肌肉以暴露头骨。然后, 创建一个小毛刺孔5毫米侧向和1毫米后向 bregma18 , 以缓解多普勒流量计铅笔探针的尖端位置。</li>
	<li>将大鼠从俯卧位置转到仰卧位, 然后将激光笔探头插入先前钻孔的颅骨盲孔, 监测区域脑血流 (局部脑血)。</li>
	<li>记录每个动物的基线局部脑血, 并将其视为100%。值得注意的是, 每当检测到80% 局部脑血的减少时, 大脑中动脉闭塞 (MCAO) 就开始了19, 20.</li>
</ol>2. 诱导灶性脑缺血
<ol><li>用 betadine 溶液和70% 乙醇对颈部进行剃须, 并对皮肤进行消毒。在手术部位注射0.2 毫升0.5% 布比卡因镇痛17。</li>
	<li>在颈部的腹面上做一个2厘米长的手术切口, 以进入左颈总动脉 (LCCA)。从相邻筋膜和迷走神经中分离 LCCA, 以获得颈外动脉 (ECA) 和颈内动脉的分岔。</li>
	<li>结扎永久 LCCA 或非洲经委会使用5-0 丝缝合和解剖 ICA 自由的水平的翼腭动脉。</li>
	<li>松散地放置在 LCCA 周围的5-0 丝缝合领带, 然后临时 ICA 钳与血管微夹。</li>
	<li>在先前放置松散的领带之前, 在 LCCA 上做一个小切口, 然后在 ICA 的腔内插入4-0 的硅涂层尼龙缝线, 并收紧 LCCA 周围的缝合, 以确保尼龙缝合, 防止血液泄漏。</li>
	<li>从 ICA 中取出血管微夹。然后, 提前一个硅涂层腔内灯丝, 直到观察到明显下降的局部脑血, 表明闭塞的 MCA 起源。在缺血性期结束时 (90 分钟), 通过取出腔内缝合21开始再灌注。</li>
</ol>3. 颈静脉插管和埃文斯蓝 (EB) 注射液
<ol><li>在颈部向中线的左侧做一个1厘米的纵切口, 然后直截了当地解剖掉浅筋膜, 以进入左颈静脉的外侧分支 (轻型货车)。然后, 用5-0 丝缝线永久结扎轻型货车的颅骨末端。在静脉周围松散地放置两条领带, 然后用血管微钳暂时夹住轻型货车的心脏末端。</li>
	<li>在两条缝线之间, 在轻型货车上做一个小切口, 然后将血气血清填充导管插入轻型货车的腔内, 并将其推进约10毫米. 随后, 拧紧静脉周围的连字, 以确保导管的安全并防止出血。注射少量的血清以避免血管塌陷。</li>
	<li>在再灌注期开始时, 在5分钟内缓慢注射 eb 染料 (1 毫升/千克 2% eb 溶液生理盐水)。随后, 通过注射0.5 毫升生理盐水冲洗套管, 从静脉12,22取出注射套管。</li>
	<li>最后, 缝合颈部和头部切口, 注射0.05 毫克/千克丁丙诺啡腹腔 (IP) 和重复注射每6-8 小时在术后镇痛17。</li>
	<li>注入5毫升预热生理盐水 (IP), 以提供恢复笼17中的水合。</li>
	<li>将动物放在装有温度和空调控制的特殊恢复笼中, 并监测动物从麻醉中恢复的情况。</li>
</ol>4. 血脑屏障通透性评估
<ol><li>再灌注24小时后, 用硫喷妥钠深麻醉动物。然后, 通过在胸骨下做一个小洞来打开胸腔。</li>
	<li>在右心房开一个小开口, 在 110 mmHg 的压力下, 通过左心室注射250毫升预热的0.9% 生理盐水 (37 ˚C), 将 EB 从循环中冲洗出去, 直到从心房的正常生理盐水出口变为无色的15。</li>
	<li>立即将大脑从头骨中取出, 并将其置于脑基质中。解剖嗅球和小脑, 然后使用干净的剃刀刀片, 从左 (损伤) 沿中线分开大脑的右半球。</li>
	<li>在2.5 毫升磷酸盐缓冲盐水中分别称量每个半球并融汇它们, 并将其与2.5 毫升的乙酸酸 (60%) 混合, 使用涡旋机进行2分钟。</li>
	<li>离心机的大脑样本为30分钟, 在 1322 x g, 并允许样品冷却在4°c 冰箱10分钟。</li>
	<li>使用上清液估计在 610nm23 的分光光度计的 EB 吸光度。</li>
	<li>根据标准曲线计算 EB 浓度, 并将结果表达为脑组织的µg/g。</li>
</ol>5. 电子束标准曲线的制作
<ol><li>在5毫升磷酸盐缓冲盐水中制备10个 eb 的样品溶液, 其浓度为 eb 的1-10 µg。</li>
	<li>使用分光光度计测量每样样品的吸光度值 610 nm。</li>
	<li>使用电子表格制作散点图, 并在 X 轴上显示 EB 浓度和 Y 轴上的吸光度值 (图 1)。</li>
	<li>用方程定义曲线的线性趋势线。然后, 用它来获得样品的 EB 浓度。</li>
	<li>报告最终结果的 EB 渗出作为µg/g 的脑组织重量。</li>
</ol>6. 假操作
<ol><li>对假手术组的大鼠进行相同的手术处理 (包括在颈部和 EB 注射液中进行切口), 但排除 MCAO。</li>
</ol>

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+induced+hypertension+improves+blood+flow+and+oxygen+metabolism+in+transient+focal+cerebral+ischemia.">Mild induced hypertension improves blood flow and oxygen metabolism in transient focal cerebral ischemia.</a> Stroke. 39 (5), 1548-1555 (2008).</li><li>Bottiger, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cerebral+ischemia+due+to+cardiocirculatory+arrest+in+mice+causes+neuronal+degeneration+and+early+induction+of+transcription+factor+genes+in+the+hippocampus.">Global cerebral ischemia due to cardiocirculatory arrest in mice causes neuronal degeneration and early induction of transcription factor genes in the hippocampus.</a> Brain Res Mol Brain Res. 65 (2), 135-142 (1999).</li></ol>

作者没有什么可透露的。

迄今为止, 各种方法, 如显影和检测放射性示踪剂24,25, 免疫荧光显微镜26,27, EB 渗出技术20, 23已被用来评估血脑屏障损伤。EB 染料强烈能够绑定到血清白蛋白, 并作为追踪器, 以检测血管渗漏和量化的 BBB 击穿11,28,29。EB 渗出技术作为一种高度接受和可靠的方法, 直接评估了脑屏障的完整性, 其影响因素包括缺血性中风。
在体内评估脑屏障允许研究人员研究可能的病理生理机制的缺血诱发血管源性水肿和寻找新的治疗干预。此模型不需要特殊的设备, 并且可以产生可靠的结果, 在实验中成功率高 (超过 80%)13,20。通过对脑组织的直接接触, 这个模型能够高度准确地评估血屏障完整性, 但限于长期研究。
缺血性脑卒中引起的脑屏障病理改变有三阶段: 急性 (小时内), 亚急性 (小时至天), 慢性 (天至月)30,31。显然, 早期的治疗干预在急性病理阶段产生有价值的保护作用。EB 剂量和注射时间点是获得可靠结果的两个关键参数, 由于脑屏障在缺血性侮辱后的动态性质。因此, 使用适当剂量 (生理盐水中的1毫克/千克 2% eb 溶液) 在再灌注期开始后, 通过静脉套管缓慢注射 EB 染料是一个重要因素, 允许研究脑卒中早期病理生理学的变化。.
本文介绍了几种实验方法对缺血性中风的研究。在这个实验模型中, 我们使用 MCAO 与腔内灯丝方法, 创建类似人类中风的条件21,32。该技术简单可靠;然而, 它的执行需要考虑到一些技术要点, 以进一步提高技术的性能, 确保其准确性。在手术期间, 体温应保持在生理范围内, 而血压和血气必须监测33,34,35。用激光多普勒流量计对局部脑血进行恒定记录, 使用合适的硅胶包覆灯丝, 不仅能提高 MCAO 成功率, 而且能降低死亡率。

血管源性脑水肿由于血脑屏障 (BBB) 中断仍然是缺血性中风的重要并发症, 也是中风患者存活率的主要决定因素1, 2.血脑屏障 (BBB) 由脑毛细血管内皮细胞 (BCECs) 形成, 由不同的神经血管成分组成 (例如, BCECs、毛细血管、星形胶质和神经细胞之间的紧密连接3), 提供了一种中枢神经系统 (CNS) 与外周血循环之间的专门和动态接口4,5。诸如缺血再灌注损伤等侮辱可能扰乱脑屏障功能完整性, 导致循环白细胞随后渗入大脑实质, 最终引发脑部炎症和原发性脑损伤。6,7. 需要动物模型来精确检测中风发生后的脑屏障功能障碍。这些模型对于研究潜在的病理生理学机制和引入新的神经保护策略具有重要意义。体外基于细胞培养的脑屏障模型已得到高度发展, 并用于生理病理的分子研究(bbb) 8, 9, 10.然而, 在这方面,在体内动物模型, 它产生的脑屏障的缺血性损伤类似于人类的临床条件, 也是非常值得的。定量检测埃文斯蓝 (EB) 的渗出是一种公认和敏感的技术, 已用于评估脑屏障完整性和功能的神经退行性疾病, 包括缺血性中风11,12,13,14. 该方法成本效益高, 可行, 重现性好, 完全适用于任何实验实验室。它的实现不需要先进的设备, 如放射性示踪剂15或磁共振成像 (MRI)16, 这是其他方法的先决条件。本文对缺血性脑卒中大鼠模型中 EB 渗出进行了脑屏障的基本技术过程的综合演示。

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an in vivo assessment of blood-brain barrier disruption in a rat model of ischemic stroke

缺血性中风导致血管源性脑水肿和随后的原发性脑损伤, 这是通过破坏血脑屏障 (BBB) 介导的。建立了诱导缺血性中风大鼠, 并作为体内模型对脑屏障功能完整性进行了研究。分光光度法检测脑标本中的埃文斯蓝 (EB), 可以为新的治疗方式的研究和开发提供可靠的依据。这种方法产生可重现的结果, 并适用于任何实验室, 不需要特殊设备。在此, 我们提出了一个可视化的技术指南, 以检测的 EB 渗出后, 诱导的缺血性中风的大鼠。

右半球 EB 水平与假手术大鼠左半球无显著性差异 (分别为 1.06 0.1 µg/克和 1.1 @ 0.09 µg/克)。如图 2 a-2 b所示, 在缺血性大鼠的左半球, 诱导短暂缺血 (90 分钟缺血/24 小时再灌注) 导致 EB 水平显著差异 (10.41 @ 0.84 µg/g, p < 0.001), 与各自半球在假操作的老鼠。总的来说, 这些发现表明, 在正常情况下, eb 不能轻易地越过脑屏障进入大脑实质和脑缺血性侮辱诱发 EB 渗出通过增强通透性的屏障 (图2A 和 2B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57156/57156fig1.jpg"> 图 1: 标准曲线用于从吸光度值中确定 EB 浓度.<a href="/files/ftp_upload/57156/57156fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57156/57156fig2.jpg"> 图 2: 在缺血性中风后24小时内 EB 对脑屏障中断的评估.假手术和缺血性动物大脑的照片 (A)。在脑组织 (蓝色) 中 EB 渗出的强度来自损伤半球的血屏障紊乱程度。在假手术和缺血性动物 (B) (n=6, *p< 0.001 与假组左半球比较, p< 0.001 相比, 从左 (损伤) 和右脑半球制备的 EB 浓度比较同组的同侧半球)。<a href="/files/ftp_upload/57156/57156fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke at JoVE.com

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

本程序的总体目标是为在体内评估缺血性中风模型大鼠血脑屏障中断提供一种高度可重复的技术。

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

an-vivo-assessment-blood-brain-barrier-disruption-rat-model-ischemic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

11.3K 次观看.  Ardabil University of Medical Sciences. 本程序的总体目标是为在体内评估缺血性中风模型大鼠血脑屏障中断提供一种高度可重复的技术。

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

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

代的永生化小鼠脑微血管内皮细胞株作为<em在体外</em血脑屏障型号

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

科研

神经科学

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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 ...

Optimized Management of Endovascular Treatment for Acute Ischemic Stroke

This manuscript describes a streamlined protocol for the management of patients with acute ischemic stroke, which aims at the minimization of time from hospital admission to reperfusion. Rapid restoration of cerebral blood flow is essential for the outcomes of patients with acute ischemic stroke. Endovascular treatment (EVT) has become the standard of care to accomplish this in patients with acute stroke due to large vessel occlusion (LVO). To achieve reperfusion of ischemic brain regions as fast as possible, all in-hospital time delays have to be carefully avoided. Therefore, management of patients with acute ischemic stroke was optimized with an interdisciplinary standard operating procedure (SOP). Stroke neurologists, diagnostic as well as interventional neuroradiologists, and anesthesiologists streamlined all necessary processes from patient admission and diagnosis to EVT of eligible patients. Target times for every step were established. Actually achieved times were prospectively recorded along with clinical data and imaging scores for all endovascularly treated stroke patients. These data were regularly analyzed and discussed in interdisciplinary team meetings. Potential issues were evaluated and all staff involved was trained to adhere to the SOP. This streamlined patient management approach and enhanced interdisciplinary collaboration reduced time from patient admission to reperfusion significantly and was accompanied by a beneficial effect on clinical outcomes.

The outcome of patients with acute ischemic stroke depends on swift restoration of cerebral blood flow. This protocol aims at optimizing the management of such patients by minimizing peri-procedural timings and rendering the time from hospital admission to reperfusion as short as possible.

急性缺血性中风血管内治疗的优化管理

This manuscript describes a streamlined protocol for the management of patients with acute ischemic stroke, which aims at the minimization of time from ...

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 ...

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 ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

评估大鼠急性中风后脑恢复的临床前模型

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

在鼠模型中使用聚焦超声波打开位置特定血脑屏障开口的台面方法

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

测量一组啮齿动物脑样本中的中风后脑水肿、梗塞区和血脑屏障破裂

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

生态HIV脑感染的老鼠模型

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...