这项工作是由国家健康和医学研究委员会澳大利亚，猎人医学研究所，以及纽卡斯尔大学支持的研究经费。

所有的程序都来自纽卡斯尔大学动物护理和伦理委员会，并按照相关指引及法规，包括新南威尔士州动物研究法，新南威尔士州动物研究法规和实务的澳大利亚守则进行审批保养和科学用途的动物。 1。脑组织解剖<ol><li>断头牺牲12周岁雄性大鼠。老鼠可以是由惊人的和断头处死，或者可以先异氟醚麻醉（5％感应，1.5-2％维护）在70％的N 2和30％O 2，然后斩首。 </li><li>从头骨尽可能迅速取出大脑（理想情况下，这应该是在不到2分钟执行）。 </li><li>通过徒手剥离取出皮层。解剖应在尽可能短的时间来执行，用尽可能少的伤害到大脑尽可能以迈ntain切片的完整性。 </li><li>为了除去任何残余的皮肤，皮毛，血液等 ，浸入大脑中的37℃的Krebs缓冲液（118 mM的氯化钠，4.7 mM的氯化钾，1.3 mM的氯化钙少量（3-5毫升）中， 1.2mM的磷酸二氢钾，1.2mM的硫酸镁，25mM的碳酸氢钠，11.7毫摩尔葡萄糖，0.03 mM的乙二胺四乙酸二钠（EDTA），用95％氧/ 5％二氧化碳（卡波金），pH 7.4）中。 </li></ol> 2。 Microslices的制备<ol><li>将大脑的McIlwain斩波器上的滤纸两片已放置和蘸有温暖的克雷布斯缓冲阶段。保持大脑和蘸有温暖的Krebs缓冲液的滤纸上，但没有那么湿，有可能会导致大脑左右滑动的纸张的表面上的游离液体。 </li><li>切片脑入250微米的冠状切片。作为营养物质，氧气和废料次在通常的血液供给交换用的流体围绕所述组织中分离的神经组织交换，切片的尺寸应不超过400微米的厚度，以便有足够的氧合和物质交换发生。 </li><li>转动McIlwain阶段90°。 </li><li>大脑切片成250微米的矢状切片。本文中，这些脑切片（250微米×250微米的切片长度可变的取决于组织的厚度）被称为microslices。 </li><li>将microslices进入圆底管​​10-15毫升37℃的Krebs缓冲液（所用Krebs缓冲液的量将取决于组织的量）。 </li></ol> 3。 Microslices的平衡<ol><li>轻轻搅拌直至个人microslices暂停，然后让它们在重力作用下沉降。 </li><li>小心取出上清液，这将是阴天，由于悬浮细胞碎片，并加入10 ml新鲜的37℃Krebs缓冲液的试管中。 </li><li>重复此洗涤步骤4次，这将导致在去除大部分碎片的。 </li><li>在此之后，平衡的microslices在37℃轻轻摇动/反转，并更改Krebs缓冲液，每15分钟1小时总共。 </li><li>这时，加入2ml新鲜的37℃Krebs缓冲液，并转移15-20 microslices（150微升microslice暂停），以平底聚苯乙烯使用一个额外的大口径枪头平滑边缘（这可以通过创建5毫升管切〜2 mm从P1000的尖底）。均匀分布的microslices在管中的单个层的基极，以使每个microslice不进一步比从含氧气氛几毫米。 </li></ol> 4。兴奋刺激<ol><li>孵育microslices在37℃，并不断通过CARBOGEN在150微升microslice悬浮液的表面上，用气相线即位置编只是microslice悬挂（避免机械破碎）的表面之上。 </li><li>治疗microslices与任何150微升5毫米谷氨酸+100μM甘氨酸在Krebs缓冲液（兴奋性刺激：终浓度为2.5 mM的谷氨酸+50μM甘氨酸）或150微升克雷布斯单独缓冲液（控制非刺激）。各种刺激可以用在该步骤中（包括，但不限于，AMPA，NMDA +甘氨酸，氯化钾去极化，和氧/葡萄糖剥夺）。 </li><li>取出培养溶液在不同时间刺激后（0，30，60，90，120，和300秒），并将其与300微升冰冷的匀浆缓冲液（30毫摩尔Tris，pH值7.4，1mM的乙二醇代替四乙酸，4毫摩尔EDTA，100μM钼酸铵，5毫焦磷酸钠，25mM的氟化钠，1mM的原钒酸钠，1mM的phenylmethanesulfonylfluoride，完全蛋白酶抑制剂混合物）。 </li><li>均质microslices在冰上，用的是玻璃 - 聚四氟乙烯Dounce匀浆（20杆; 700转）。 </li><li>店铺匀浆在-80℃下，各种信号转导分子，死亡分子等可以通过蛋白印迹分析来测定。 </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+of+cAMP/PKA+and+MAPK+signaling+in+neuronal+cells+is+dependent+on+developmental+stage.">Coupling of cAMP/PKA and MAPK signaling in neuronal cells is dependent on developmental stage.</a> Exp. Neurol. 169 (1), 44-55 (2001).</li><li>Kharlamov, E., Cagnoli, C. M., Atabay, C., Ikonomovic, S., Grayson, D. R., Manev, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+effect+of+protein+synthesis+inhibitors+on+potassium+deficiency-induced+apoptotic+cell+death+in+immature+and+mature+neuronal+cultures.">Opposite effect of protein synthesis inhibitors on potassium deficiency-induced apoptotic cell death in immature and mature neuronal cultures.</a> J. Neurochem. 65 (3), 1395-1398 (1995).</li><li>Kristensen, B. W., Noraberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+excitotoxic+profiles+of+ATPA,+AMPA,+KA+and+NMDA+in+organotypic+hippocampal+slice+cultures.">Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures.</a> Brain Res. 917 (1), 21-44 (2001).</li><li>Lecrux, C., 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=Spontaneously+hypertensive+rats+are+highly+vulnerable+to+AMPA-induced+brain+lesions.">Spontaneously hypertensive rats are highly vulnerable to AMPA-induced brain lesions.</a> Stroke. 38, 3007-3015 (2007).</li><li>Sattler, R., Charlton, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3H]MK-801+binding+in+immature+and+mature+chicken+forebrain.">3H]MK-801 binding in immature and mature chicken forebrain.</a> Neurosci. Lett. 134 (1), 83-87 (1991).</li><li>Kavanagh, J. M., Bunn, S. J., Boyd, T. L., Rostas, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+glutamate+receptor+stimulated+inositol+phospholipid+metabolism+and+45Ca(2+)-accumulation+in+posthatch+chicken+forebrain.">Developmental changes in glutamate receptor stimulated inositol phospholipid metabolism and 45Ca(2+)-accumulation in posthatch chicken forebrain.</a> Neurosci. Lett. 194 (3), 161-164 (1995).</li><li>Sharps, E. S., McCarl, R. 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作者宣称，他们有兴趣就任何本手稿中开展的工作没有冲突。

本文中， 体外技术microslices可用于检查涉及兴奋性中毒和缺血介导的细胞死亡在完整成熟的脑组织的分子机制的生成进行说明。该技术生产的活组织（ 图1-3），即代谢类似于较大的器官切片15。此外，这种microslice模式紧密对应观察下面的体内 10介导的兴奋性毒性脑损伤的反应。 90秒脑 microslices与AMPA，NMDA或谷氨酸体外刺激诱 ​​导多种分子...

中风的最常见的形式是缺血性中风，当脑血管变得闭塞而发生。该组织缺血导致从停止血流引起膜的去极化普遍，兴奋性神经递质的释放，细胞内钙，从而导致细胞死亡的活性的持续升高的途径1。这个过程已经被称为“兴奋性中毒”，并且是参与神经元死亡的一个常见的途径由多种病症，包括中风2制备。参与兴奋性毒性及其他神经细胞死亡的级联信号通路的抑制是一个有吸引力的方式来限制中风后神经元损伤。 确定在兴奋性毒性和缺血性中风的确切分子机制可以用整体动物系统时会很困难。因此，初级胚胎和“神经元样”（ 例如 neuroblasto毫安和腺癌永生化线）细胞培养系统经常被使用。这些模型的主要优点是它们易于操作的，相对低成本，和细胞死亡可容易地测量和定量。然而，信号通路可以通过使用3,4的培养条件，以及未成熟的神经元改变和永生线可以表达不同的受体和较成熟的大脑5-8信号分子。此外，培养的神经元只允许一个小区类型的检测（或2，如果一个共存系统使用的），而完整的脑组织是异质的，含有多种与彼此交互的细胞类型。器官切片培养系统（薄植脑组织）也被使用，并且这些模型使细胞的异质群体的研究，因为他们被发现在体内 。但是，可以从每个动物使用这种技术时获得的组织只有数量有限，切片可未进行培养，只要永生化细胞系，并且介质给长期培养可导致改变的信号转导途径和受体的切片。尽管成熟的大脑可以被用来生成器官切片，从不成熟的脑切片更适于培养，而且更普遍使用。因此，有必要开发一种模仿或代表完整成熟的大脑，是易于使用，其中神经信号传导通路可以检查模型。 本文中， 在体外 ，涉及可用于阐明参与细胞死亡的分子机制下列的兴奋毒性或缺血性损伤完好的神经组织的技术进行说明。这种技术减少了要执行的实验所需的动物数量，是可重现的，并产生其行为在代谢上类似的方式来放大器官切片活组织。此外，神经回路，细胞相互作用，和突触后共同mpartment部分仍然完好。用生理缓冲使细胞膜“重新封装”，并且使细胞恢复其原来的膜电阻9。这种脑片模型能够忠实地模仿观察到下列介导的兴奋性毒性脑损伤10的反应，并且可以用来检查涉及中风的分子机制。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Guillotine</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to decapitate animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical equipment</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Forceps, scissors, tweezers, etc, for brain removal and dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">McIlwain chopper</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Used to generate 100-400 &#956;m brain sections.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>McIlwain choppers are manufactured/distributed by a range of companies including Mickle Engineering, Harvard Apparatus, Campden Instruments and Ted Pella.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Round bottom plastic tubes</td>
			<td style="width:71px;">Greiner</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>For keeping tissue at 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aerating apparatus</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Used to keep microslices in a humidified, oxygenated environment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Flat bottomed polystyrene tubes</td>
			<td style="width:71px;">Nunc</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dounce Homogenizer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

excitotoxic stimulation of brain microslices as an in vitro model of stroke

检查涉及神经病理症状的分子机制，如缺血性中风，可以使用整个动物系统时是困难的。因此，伯或“神经元样”细胞培养系统通常利用。虽然这些系统相对容易的工作，并且是有用的模型系统中各种功能的结果（如细胞死亡）可以很容易量化的研究成果和途径培养未成熟的神经元（如兴奋毒性介导的细胞死亡途径）不一定是相同的成熟大脑观察到，或者在完整的组织。因此，有必要开发模型，其中在成熟的神经组织的细胞机制可以被检查。我们已经开发出一种体外技术，可以用于研究各种在完好的神经组织分子途径。本文描述的技术利用大鼠皮质组织，但该技术可以适用于使用组织从多种物种（如小鼠，兔，豚鼠，鸡）或脑区域（例如，海马，纹状体， 等等 ）。此外，各种刺激/治疗方法可以被使用（例如，兴奋毒性，抑制剂的施用等 ）。总之，本文所述的脑切片模型可用于研究多种参与兴奋性毒性介导的脑损伤的分子机制。

使用此过程产生Microslices是可行的，并有多种物种（例如大鼠，小鼠和鸡），可用于产生microslices。已被利用的可行性的三个独立的措施：呼吸速率（ 图1），腺嘌呤核苷酸的比例（ 图2），和组织的钾含量（ 图3）。采用这些措施，它已经证明，microslices保持活力至少2小时后生成。 脑microslices可以用来检查各种信号转导分子，下列各种刺?...

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Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

我们已开发了可用于检查涉及兴奋性毒性介导的脑损伤的分子机制的脑切片的模型。这种技术产生可行的成熟的脑组织，并减少所需的实验动物数量，同时保持神经回路，细胞相互作用，和突触后车厢部分完好无损。

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

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Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

8.4K 次观看.  The University of Newcastle. 我们已开发了可用于检查涉及兴奋性毒性介导的脑损伤的分子机制的脑切片的模型。这种技术产生可行的成熟的脑组织，并减少所需的实验动物数量，同时保持神经回路，细胞相互作用，和突触后车厢部分完好无损。 

视频: Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

双侧颈总动脉闭塞足够的预处理刺激诱导局灶性脑缺血早期缺血耐受

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

科研

医学

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

蛛网膜下腔出血的小鼠模型

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

作为脑微血管内皮细胞（CEND）的伸展<em&gt;体外</em创伤性脑损伤模型血脑屏障

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

设置了一个<em&gt;体外</em大鼠血脑屏障&gt;模型（BBB）：在BBB抗渗性和受体介导的运输为中心

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

缺氧缺糖再给氧作为<em&gt;在体外</em&gt;缺血再灌注损伤模型的研究血脑屏障功能障碍

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations over time in the blood-oxygenation level dependent signal. rs-fcMRI has been applied extensively to identify abnormalities in brain connectivity in different neurologic and psychiatric diseases. However, the relationship among rs-fcMRI connectivity abnormalities, brain electrophysiology and disease state is unknown, in part because the causal significance of alterations in functional connectivity in disease pathophysiology has not been established. Transcranial Magnetic Stimulation (TMS) is a technique that uses electromagnetic induction to noninvasively produce focal changes in cortical activity. When combined with electroencephalography (EEG), TMS can be used to assess the brain's response to external perturbations. Here we provide a protocol for combining rs-fcMRI, TMS and EEG to assess the physiologic significance of alterations in functional connectivity in patients with neuropsychiatric disease. We provide representative results from a previously published study in which rs-fcMRI was used to identify regions with abnormal connectivity in patients with epilepsy due to a malformation of cortical development, periventricular nodular heterotopia (PNH). Stimulation in patients with epilepsy resulted in abnormal TMS-evoked EEG activity relative to stimulation of the same sites in matched healthy control patients, with an abnormal increase in the late component of the TMS-evoked potential, consistent with cortical hyperexcitability. This abnormality was specific to regions with abnormal resting-state functional connectivity. Electrical source analysis in a subject with previously recorded seizures demonstrated that the origin of the abnormal TMS-evoked activity co-localized with the seizure-onset zone, suggesting the presence of an epileptogenic circuit. These results demonstrate how rs-fcMRI, TMS and EEG can be utilized together to identify and understand the physiological significance of abnormal brain connectivity in human diseases.

Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity.

评价中的连接有关的大脑兴奋患者癫痫多式联运Imaging-和激励为基础的方法

Resting-state functional connectivity MRI (rs-fcMRI) is a technique that identifies connectivity between different brain regions based on correlations ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Vagus Nerve Stimulation As an Adjunctive Neurostimulation Tool in Treatment-resistant Depression

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression (TRD). VNS exhibits antidepressive and stabilizing effects. This method is particularly useful as a long-term treatment, in which up to two-thirds of patients respond. The vagus nerve stimulator is positioned on the left vagus nerve during a surgical procedure and is activated telemetrically by a wand connected to a handheld computerized device. The treating physician can perform various adjustments of the vagus nerve stimulator during in-office visits (e.g., by modifying stimulation intensity or stimulation frequency) to achieve maximum therapeutic effects with low side effects. Set-up of the device usually takes several months. Typical side effects include wound infection, temporary salivation, coughing, paralysis of the vocal cords, bradycardia, or even asystole. The patient can stop the VNS by placing a magnet over the generator. The current protocol describes delivery of the specific stimulation tool and methods for adjusting the tuning parameters to achieve the best remission rates in patients with TRD.

Vagus nerve stimulation (VNS) has been shown to be effective as an adjunct treatment for treatment-resistant depression (TRD). VNS leads to antidepressive and antisuicidal effects and quality of life improvements. This protocol offers a step-by-step guide to managing and adjusting a vagus nerve stimulator for efficient treatment of TRD.

迷走神经刺激作为抗抑郁抑郁症的辅助神经刺激工具

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression ...

Updated Technique for Reliable, Easy, and Tolerated Transcranial Electrical Stimulation Including Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of brain stimulation presents several potential advantages compared to other techniques, as it is noninvasive, cost-effective, broadly deployable, and well-tolerated provided proper equipment and protocols are administered. Even though tDCS is apparently simple to perform, correct administration of the tDCS session, especially the electrode positioning and preparation, is vital for ensuring reproducibility and tolerability. The electrode positioning and preparation steps are traditionally also the most time consuming and error-prone. To address these challenges, modern tDCS techniques, using fixed-position headgear and pre-assembled sponge electrodes, reduce complexity and setup time while also ensuring that the electrodes are consistently placed as intended. These modern tDCS methods present advantages for research, clinic, and remote-supervised (at home) settings. This article provides a comprehensive step-by-step guide for administering a tDCS session using fixed-position headgear and pre-assembled sponge electrodes. This guide demonstrates tDCS using commonly applied montages intended for motor cortex and dorsolateral prefrontal cortex (DLPFC) stimulation. As described, selection of the head size and montage-specific headgear automates electrode positioning. Fully assembled pre-saturated snap-electrodes are simply affixed to the set position snap-connectors on the headgear. The modern tDCS method is shown to reduce setup time and reduce errors for both novice and expert operators. The methods outlined in this article can be adapted to different applications of tDCS as well as other forms of transcranial electrical stimulation (tES) such as transcranial alternating current stimulation (tACS) and transcranial random noise stimulation (tRNS). However, since tES is application specific, as appropriate, any methods recipe is customized to accommodate subject, indication, environment, and outcome specific features.

When administering transcranial direct current stimulation (tDCS), reproducible electrode preparation and placement are vital for a tolerated and effective session. The purpose of this article is to demonstrate updated modern setup procedures for the administration of tDCS and related transcranial electrical stimulation techniques, such as transcranial alternating current stimulation (tACS).

用于可靠、简单和耐受的颅内电刺激（包括颅内直流刺激）的更新技术

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of ...