SN和MDD从NIH K08 NS083740和神经病学的加州大学洛杉矶分校部门的支持。 AJG承认由仪博士和谢尔登·阿德尔森G.医学研究基金会和拉里·希尔布鲁姆L.基金会的支持。九龙十分感谢美国心脏协会14BFSC17760005 ASA-Bugher卒中中心的支持。 ILL，EGS和STC是由美国国立卫生研究院R01 NS071481支持。 JDH确认来自美国国立卫生研究院K08 NS083740支持。

在这个协议中使用的动物是按照由加州大学洛杉矶分校的动物护理和使用委员会的批准大学的程序进行。 注:确定目标人群小鼠开始。在以前的研究中，只有雄性野生型C57 / BL6小鼠已被使用，但是各种转基因或基因敲除小鼠也可以使用。注意，立体定向坐标基于C57 / BL6解剖。建议每个用户最初验证中风白质的定位。 1.白质行程感应 - 内侧斜角方法<ol><li>通过使用0.5毫米毛细管使得远端直径为15-25微米11之间制备拉玻璃吸管开始。 </li><li>在无菌的0.9％生理盐水27.4毫克/毫升（130μM） - 制备L-NiO的无菌10微升等分试样（（1）-iminoethyl -L-鸟氨酸盐酸N（5））。 </li><li>预填拉玻璃吸管与由固定用玻璃吸管的小体积的L-仁王（2-5微升）至管连接到真空管线。躺在工作台上吸管平并插入拔出端插入L-仁王解决方案。 <ol><li>直到吸管0.5毫米部的至少2mm填充应用真空。关闭真空和撤回移液器。将它放在一旁，直到步骤1.12。 </li></ol></li><li>鼠标放置在感应腔和诱导使用标准的32％异氟烷鼠标麻醉通过蒸发器流入（5升/分钟吸入用5升/分钟的氧气和0.5升/分钟N 2），持续1分钟或直至深麻醉。鼠标转移到配备有立体显微镜立体定位装置。提供一种使用32％异氟烷维持麻醉通过蒸发器流入（2L /分吸入用5升/分钟的氧气和0.5升/分钟N 2）和一鼻锥体。检查麻醉深度与脚趾捏。 </li><li>注射臂调整到36°。 </li><li>阿菲XA拉玻璃吸管持有人一个低量的压力喷射系统的末端，并将其连接到立体设置的注入手臂。 </li><li>涂抹麻醉动物用凡士林和地点人工泪液软膏超过两只眼睛胡须。通过将无菌悬垂在动物的头用5-10厘米开幕的头准备一个无菌手术领域。剃皮毛覆颅骨准备aspetic手术的表面。清洁交替优碘和70％酒精棉签头皮。 </li><li>请用无菌剪刀罚款1.5厘米的头皮正中切口，显露颅骨表面。干后用消毒棉签头骨和1-3X的放大倍率用显微镜立体，用无菌微点工具可以删除任何覆盖骨膜组织。 </li><li>标记为前囟门用细点标记的参考点。 </li><li>使用无菌细STIP手术钻头钻2毫米ellipitical开颅手术0;在后方前囟门开始，向前延伸刚出中线。去除骨碎片和上覆的软组织，以便大脑皮层可以可视化。 </li><li>保持手术视野和皮质表面湿润通过间歇应用无菌生理盐水滴。 </li><li>贴上拉玻璃吸管立体定向仪的注射器手臂。对准前囟吸管的远端和零的立体定向坐标。 </li><li>前进移液器到第一前/后（A / P）和内侧/外侧（M / L） 表1坐标提供。 </li><li>推进吸管皮质表面和零背/腹（D / V）的测量。 </li><li>慢慢通过吸管进入脑，直到达到表1的第一D / V坐标。 </li><li>使用低容量压力喷射系统20毫秒脉冲设置为20磅，注入100 NL L-仁王的入脑，并等待5分钟，以防止反流了吸管轨道。 <ol><li>在立体定向显微镜的目镜和3X的倍率使用校准掩模版。 </li><li>因此，置换共有0.100毫米3（0.5毫米的长度在0.5毫米直径的移液管中，对应于100 NL）从拉玻璃吸管为每组坐标。通过使用划板，测量和标准化，因为每个设置取决于所使用的放大率和体重秤。 </li><li>对于每一个在注射过程中准确体积测量，接近与显微镜倾斜移液器从侧面使空气流体弯月面具有矢状图。弯液面应该出现在这两个吸移管的内壁和外壁的同一焦平面。 </li></ol></li><li>慢慢抽出吸管和重复步骤1.13-1.16.3在第二和第三组在表1中提供的坐标。 </li><li>最后一次注射后，取出吸管，并放置足够的骨蜡填补了开颅手术部位。一个pproximate头皮伤口的边缘，并与皮肤粘合剂结合。 </li><li>注入0.1毫升0.5％麻卡因成使用无菌​​地下30针，以防止以防止与头皮切口相关联的局部疼痛的伤口边缘。 </li><li>返回动物壳体和供应术后抗生素（0.48毫克/毫升复方新诺明，或0.5毫克/毫升左氧氟沙星）的饮用水5天。 </li></ol> 2.白质行程感应 - 后路斜角方法<ol><li>执行步骤1.1-1.12为在内侧倾斜的方式协议中，除了调整立体设置面向从前到后45度的注入手臂。 </li><li>前进移液器向第一A / P和M / L的坐标在表2中提供。 </li><li>完成剩余的步骤1.14-1.20作为在横向成角度的方法的协议。 </li></ol> 3.逆行神经标签<ol><li>制备无菌的10在0.9％的生理盐水54.8毫克/毫升的L-仁王的微升等分试样。 </li><li>制备20％Fluororuby（或20％的生物素化葡聚糖胺或2％荧光金）的0.9％生理盐水的无菌等分试样。 </li><li>稀释一起1:1 27.4毫克/毫升的L- NiO和10％Fluororuby的最终浓度。 </li><li>步骤1.3-1.23执行行程协议如上。 </li><li>如前所述8灌注固定，cryosectioning和显微可视化组织切片的原生荧光示踪剂。 </li></ol> 4.组织处理的免疫<ol><li>在适当的中风后间隔时间从3小时到中风后14天内，通过安乐死过量的异氟醚或当地IACUC小鼠批准的程序。 </li><li>使用倾斜的剪刀打开胸腔，并插入一个23政蝴蝶针进入左心室。 </li><li>放置在用细剪刀，以允许灌注流体流出轨道右心房的小切口。 </li><li>在室温为10毫升/分钟的速率用30-40毫升冷的磷酸盐缓冲盐水后30-40毫升冷的4％多聚甲醛的Transcardially灌注。 </li><li>斩首鼠标和使用无菌剪刀向后打开颅骨取出大脑，然后轻轻地用刮勺除去覆颅骨，将脑入冷的4％PFA中24小时，然后转移到30％蔗糖的PBS中48小时。 </li><li>制备40使用低温恒温器微米浮动切片，并如前所述6-8执行抗体的处理。在这项研究中，使用下列抗体:兔抗神经丝200（1:500稀释度）;兔抗波形蛋白（1:500）;山羊抗GFAP（1:500）;兔抗伊巴-1（1:1000）。 </li></ol> 5.组织处理的蛋白质或RNA分析<ol><li>在适当的中风后间隔时间从3小时到中风后14天，异氟醚通过安乐死过量或当地IACUC批准的程序。 </li><li>斩首用无菌剪刀向后打开颅骨鼠标和取出大脑，然后轻轻地用锅铲除去覆头骨。 </li><li>在大脑的前面插入一个无菌4毫米刮刀切断嗅球和视神经。轻轻抬起脑出颅骨并放入冰冷的夹层缓冲液（1×Hank氏平衡盐溶液，25mM的HEPES-KOH，pH 7.4的35毫摩尔葡萄糖，4mM的碳酸氢钠，和0.01毫克/毫升环己酰胺）。 </li><li>用脑块，并育新刀片，制成含有中风并放入冷夹层缓冲器2-3毫米的板坯。 </li><li>在解剖显微镜下，确定在所注入的半球白质底层运动皮层。在较长的脑卒中后的时间间隔，该区域可在视觉上由局灶性坏死和髓鞘苍白鉴定。 注:在早期中风后的时间间隔，L-仁王的注射1微升10％混合固绿可以让中风的视觉识别（ <strong&gt;图4A）。 </li><li>在解剖显微镜下的指导，并用新鲜的手术刀，小心解剖的白质含有行程，由任一坚牢绿标签或组织损失确定的区域。根据需要取出覆皮层和纹状体底层。 </li></ol>

<ol>
	<li>Go, A. S., 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=Heart+disease+and+stroke+statistics--2014+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2014 update: a report from the American Heart Association.</a> Circulation. 129, 28-292 (2014).</li><li>Saini, M., 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=Silent+stroke:+not+listened+to+rather+than+silent.">Silent stroke: not listened to rather than silent.</a> Stroke. 43, 3102-3104 (2012).</li><li>Koton, S., 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=Burden+and+outcome+of+prevalent+ischemic+brain+disease+in+a+national+acute+stroke+registry.">Burden and outcome of prevalent ischemic brain disease in a national acute stroke registry.</a> Stroke. 44, 3293-3297 (2013).</li><li>Jiwa, N. S., Garrard, P., Hainsworth, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+vascular+dementia+and+vascular+cognitive+impairment:+a+systematic+review.">Experimental models of vascular dementia and vascular cognitive impairment: a systematic review.</a> J Neurochem. 115, 814-828 (2010).</li><li>Sozmen, E. G., Hinman, J. D., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+that+matter:+white+matter+stroke+models.">Models that matter: white matter stroke models.</a> Neurotherapeutics. 9, 349-358 (2012).</li><li>Sozmen, E. G., Kolekar, A., Havton, L. A., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+white+matter+stroke+model+in+the+mouse:+axonal+damage,+progenitor+responses+and+MRI+correlates.">A white matter stroke model in the mouse: axonal damage, progenitor responses and MRI correlates.</a> J Neurosci Methods. 180, 261-272 (2009).</li><li>Rosenzweig, S., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+exacerbation+of+white+matter+stroke+outcomes:+a+role+for+oxidative+damage+and+inflammatory+mediators.">Age-dependent exacerbation of white matter stroke outcomes: a role for oxidative damage and inflammatory mediators.</a> Stroke. 44, 2579-2586 (2013).</li><li>Hinman, J. D., Rasband, M. N., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+the+axon+initial+segment+after+focal+cortical+and+white+matter+stroke.">Remodeling of the axon initial segment after focal cortical and white matter stroke.</a> Stroke. 44, 182-189 (2013).</li><li>McCall, T. B., Feelisch, M., Palmer, R. M., Moncada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+N-iminoethyl-L-ornithine+as+an+irreversible+inhibitor+of+nitric+oxide+synthase+in+phagocytic+cells.">Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells.</a> Brit j pharmacol. 102, 234-238 (1991).</li><li>Gadea, A., Aguirre, A., Haydar, T. F., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelin-1+regulates+oligodendrocyte+development.">Endothelin-1 regulates oligodendrocyte development.</a> J Neurosci. 29, 10047-10062 (2009).</li><li>Dean, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+(pulling)+of+needles+for+gene+delivery+by+microinjection.">Preparation (pulling) of needles for gene delivery by microinjection.</a> CSH prot. , (2006).</li><li>Hughes, P. M., 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=Focal+lesions+in+the+rat+central+nervous+system+induced+by+endothelin-1.">Focal lesions in the rat central nervous system induced by endothelin-1.</a> J. Neuropathol. Exp. Neurol. 62, 1276-1286 (2003).</li><li>Whitehead, S. N., Hachinski, V. C., Cechetto, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+a+rat+model+of+cerebral+ischemia+and+beta-amyloid+toxicity:+inflammatory+responses.">Interaction between a rat model of cerebral ischemia and beta-amyloid toxicity: inflammatory responses.</a> Stroke. 36, 107-112 (2005).</li><li>Frost, S. B., Barbay, S., Mumert, M. L., Stowe, A. M., Nudo, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+animal+model+of+capsular+infarct:+endothelin-1+injections+in+the+rat.">An animal model of capsular infarct: endothelin-1 injections in the rat.</a> Behav Brain Res. 169, 206-211 (2006).</li><li>Horie, N., 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=Mouse+model+of+focal+cerebral+ischemia+using+endothelin-1.">Mouse model of focal cerebral ischemia using endothelin-1.</a> J Neurosci Methods. 173, 286-290 (2008).</li><li>Wang, M., 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=Cognitive+deficits+and+delayed+neuronal+loss+in+a+mouse+model+of+multiple+microinfarcts.">Cognitive deficits and delayed neuronal loss in a mouse model of multiple microinfarcts.</a> Neuroscience. 32, 17948-17960 (2012).</li><li>Shih, A. Y., 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=The+smallest+stroke:+occlusion+of+one+penetrating+vessel+leads+to+infarction+and+a+cognitive+deficit.">The smallest stroke: occlusion of one penetrating vessel leads to infarction and a cognitive deficit.</a> Nat Neurosci. 16, 55-63 (2013).</li><li>Jung, K. J., 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=The+role+of+endothelin+receptor+A+during+myelination+of+developing+oligodendrocytes.">The role of endothelin receptor A during myelination of developing oligodendrocytes.</a> J Korean Med Sci. 26, 92-99 (2011).</li></ol>

已经描述了若干皮层下行程的前模型，包括内皮素-1的焦点注射进入内囊，皮层下白质和纹状体在大鼠12-14和鼠标6,15。更近的小焦点冲程模型已经利用在颈动脉16胆固醇微栓子注射和单个穿透动脉17的光化学闭塞。每个模型都有有利有弊5。目前描述的模型产生具有许多模仿人类腔隙性脑梗塞，包括轴突异常和损失，髓鞘退化，焦距坏死核心和临床赤字是最小的，并演示了较快的恢复依赖于7年龄特点的病变。鼠靶向脑白质允许多种，可以支持机制研究遗传操作的。 关键该协议的步骤包括利用立体定向精确坐标。由于鼠类白质体积小和应变变化应变，可以根据使用鼠标的年龄和应变需要的立体坐标修订。注射的体积的控制也很重要，因为这是直接相关的梗塞的大小。注射较大将产生上覆皮层和纹状体底层染指大招。 使用此处描述的方法的ET-1的焦点注射已经报道，但内皮素-1显示出具有对少突胶质细胞的分化和成熟10,18混杂中风后白质生物学研究直接旁分泌的影响。相反，L-仁王方法目标内皮细胞单独同时产生相同的病变，并消除了对参与损伤反应的细胞的任何混杂旁分泌的影响。左旋仁王不直接细胞毒性和选择素（数据未显示）泰德剂量由初步剂量递增实验确定。后倾斜的方法的开发是为了更精确地从初级运动皮质倒勾轴突产生的最大行为缺损，可以归因于白质损伤。内侧倾斜的做法也损害运动皮层的神经轴突，但更多的横向延伸，涉及到底层的轴突初级感觉皮层。 笔划病变在最初24小时的相当迅速膨胀。由七天梗塞的大小为最大，我们没有观察到显著病斑生长超出该时间。额外细胞事件和轴突变性会发生超出这个初始阶段，但如由坏死核心测定梗塞大小不会在没有任何介入的显著变化。 在行程的时间神经解剖学示踪剂的共注射标识神经元经历缺血性轴索损伤。无论使用配有IAL或后倾斜的做法，受损轴突投射神经元细胞体仍然安然无恙。这将创建一个有用的模型来研究中枢神经系统的神经细胞缺血性离断伤的效果。我们主要利用示踪剂逆行包括葡聚糖胺和荧光金，这既显示出优良的摄取中风损伤轴突。通过采用多种转基因和基因敲除小鼠品系中，本规程的用户可以调查涉及脑白质损伤中风响应和修复神经元基因的具体作用。

Stroke affecting the subcortical white matter is a common clinical entity, accounting for up to 25% of clinical strokes annually in the US 1. Ischemic damage to white matter also occurs silently at a significantly higher rate and contributes to the development of vascular dementia 2,3. Presently, patients with this form of cerebral ischemia have few, if any treatment choices. Despite the clinical importance of this disease, few clinically relevant animal models exist 4,5. 
The goal of this protocol is to produce a focal ischemic lesion within the murine white matter. This murine model of human disease allows the specific study of axonal injury response to stroke and how the cellular elements of white matter, namely oligodendrocytes and astrocytes along with axons, respond to and repair after stroke.
Previous reports have described a model of subcortical white matter stroke using endothelin-1 (ET-1) 6 that is similar to the one described here. Several key changes to the experimental protocol have been made thereby the potential uses of this model have expanded 7,8. This protocol provides a reliable and modifiable strategy to produce a focal stroke within mouse brain white matter. 
The major advantages of this model are the use of a chemical endothelial nitric oxide synthase (eNOS) inhibitor N(5)-(1)-iminoethyl-L-ornithine HCl (L-Nio) 9 with no known paracrine effects on cellular elements of white matter which had been a complication of models using endothelin-1 10. In addition, the stereotactic targeting of white matter in the mouse allows the use of any variety of transgenic or knockout strains, greatly expanding the available tools to determine the effect of stroke on brain white matter. Here, two variations on this technique are described and demonstrate some of the additional variations that can be utilized to enhance the understanding of axonal and white matter damage and repair after stroke.

<table border="0" cellpadding="0" cellspacing="0" width="725">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">L-N5-(1-Iminoethyl)ornithine, Dihydrochloride</td>
			<td style="width:204px;">Calbiochem</td>
			<td style="width:133px;">400600-20MG</td>
			<td style="width:81px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Phoenix Pharmaceutical, Inc.</td>
			<td>NDC 57319-559-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary tubes</td>
			<td>World Precision Instruments</td>
			<td>50-821-807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Picospritzer</td>
			<td>Parker Instrumentation</td>
			<td>Picospritzer II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereotactic setup</td>
			<td>Kent Scientific</td>
			<td>KSC51725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette puller</td>
			<td>KOPF</td>
			<td>Model 720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereomicroscope SZ51</td>
			<td>Olympus</td>
			<td>88-124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine scissors</td>
			<td>Fine Scientific Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Harvard Apparatus</td>
			<td>PY2 72-8547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved Forceps</td>
			<td>Harvard Apparatus</td>
			<td>PY2 72-8598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt dissection tool</td>
			<td>Fine Scientific Tools</td>
			<td>10066-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drill</td>
			<td>Dremel</td>
			<td>8220-1/28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drill bits</td>
			<td>Fine Scientific Tools</td>
			<td>19007-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vetbond</td>
			<td>3M</td>
			<td>1469SB&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Marcaine</td>
			<td>HOSPIRA</td>
			<td>NDC 0409-1610-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trimethoprim-Sulfamethaxole</td>
			<td>STI Pharmacy</td>
			<td>NDC 54879-007-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluororuby</td>
			<td>Fluorochrome Inc</td>
			<td>30mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Fisher</td>
			<td>O4042-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Fisher</td>
			<td>BP220-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Leica CM3050 S</td>
			<td>14047033518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass slides</td>
			<td>Fisher</td>
			<td>12-544-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green&#160;</td>
			<td>Sigma</td>
			<td>F7252-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ1500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">23 gauge butterfly needle</td>
			<td>Fisher</td>
			<td>14-840-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X Hank&#39;s Balanced Salt Solution</td>
			<td>Life Technologies</td>
			<td>14065056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1M HEPES-KOH, pH 7.4</td>
			<td>Affymetrix</td>
			<td>16924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyclohexamide</td>
			<td>Sigma</td>
			<td>01810</td>
			<td></td>
		</tr>
	</tbody>
</table>

a versatile murine model of subcortical white matter stroke for the study of axonal degeneration and white matter neurobiology

中风影响白质占的临床冲程演示高达25％，在费率可能5-10倍更大默默地发生，并且显著血管性痴呆的发展有助于。存在焦白质中风的几个型号，这缺乏适当的模式阻碍了参与这类中风后损伤反应和修复neurobiologic机制的理解。其他皮层下中风模型的主要限制是，它们不灶性限制梗塞的白质或已经主要在非鼠物种得到验证。这限制应用各种各样的鼠研究工具来研究白质中风的神经生物学的能力。在这里，我们提出了一个方法，为可靠的生产使用的是不可逆的eNOS抑制剂局部注射鼠白质的焦点中风。我们还提出对一般协议，包括两个独特的立体几个变化变型中，逆行神经追踪，以及新鲜组织标记和解剖，大大扩大该技术的潜在应用。这些变化允许多种方法来分析的行程这个共同的和充分研究形式neurobiologic影响。

使用提出的模型，白质底层前肢感觉运动皮层能够可靠地针对性。此化学诱导中风模型产生焦轴突和髓磷脂损失，星形胶质细胞增生，和小神经胶质细胞（ 图1），如在人类腔隙性脑梗死通常看到。通过使用三次注射，临床有用的模型建立与前肢运动任务7月初，障碍和脑组织经验，一个小而显著部分缺血的免疫，免疫和生化技术是在定量层面可行，可靠。在中风诱导后早期时间点（小时），在轴突分子组织的变化可以被检测（ 图2）。在7天，行程完成其成熟轴索损（ 图1a）的限定区。在我们的手中，这种方法产生的焦白质病变approximately 800微米水平直径和延伸沿胼胝体的前后轴约1毫米。由7天，平均总梗塞大小大约0.200毫米3和将具有椭圆形状。我们观察到约10％的变异笔触大小两种动物之间和部分，取决于其中有椭圆形的部分之间发生。在梗死面积进一步增长很少见到超过7天。 在显著神经元标记的葡聚糖胺结果的同时注射的在层5和层6具有轴突通过行程的区域突出（ 图3）的神经元细胞体的加法。不是由步骤（细针的通路）的假方面受损上方皮质神经元，进行远端轴索损害，并且可以通过包含与L仁王制备示踪物来鉴定。这种方法是使用<html> d可卒中后8展示轴突起始段的动态变化。 而受到中风的组织的区域的小尺寸限制了使用的其它常规技术，如2,3,5-氯化三苯基四唑（TTC）染色，脑白质行程区可以在新鲜组织来鉴定。增加了一个共同的染料如坚牢绿产生组织的可识别的区域，可以在解剖显微镜下解剖（ 图4）。一旦解剖这个组织可以用于与蛋白质印迹或免疫沉淀，或用于RNA分离和分析（ 图4C）蛋白分析。通过使用各种转基因小鼠线，各种创新的方法可以用来研究焦点白质行程包括细胞命运的映射研究和激光捕获显微切割（ 图4C）后，神经生物学。 帐篷"FO: - together.within页保留="1"&gt; <img alt="图1" src="/files/ftp_upload/53404/53404fig1.jpg" /> 图1:焦白质行程同时使用内侧和后路角度的途径为神经丝免疫荧光标记（A，红色）演示了行程七天后轴突损失的使用方法内侧的程度。使用后倾斜的做法，脑白质病变行程是仅高于侧脑室（B，C），针对性和显示强烈的小胶质细胞（B）和星形胶质细胞反应性（C）。两个星形胶质细胞中间丝标记，波形蛋白（红色）和胶质纤维酸性蛋白（GFAP，绿色），无一不透露行程（C）后变化的白质（纤维）星形胶质细胞的形态。比例尺= 500微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53404/53404fig1large.jpg" target="_blank">请点击此处查看大版本</a>这个数字的锡永。 <img alt="图2" src="/files/ftp_upload/53404/53404fig2.jpg" /> 图2:脑白质行程改变轴突微区组织内白质行程感应，轴突微区组织的节点上，通过测试-IV血影（红色），并在paranode标记，通过contactin相关蛋白标记的3小时（CASPR，绿），被破坏（箭头B）。对侧白质轴突显示常规节点，paranodal和juxtaparanodal组织（A和C），而同侧白质显示节点和paranodal伸长率是典型的与失去axoglial接触（B和D）轴突。比例尺=5μm以下。 <img alt="图3" src="/files/ftp_upload/53404/53404fig3.jpg" /> 图3:逆行神经Labelin摹与脑白质中风标识单个神经元与轴索损害。卒中诱导的实时荧光葡聚糖胺（红色）联合注射允许与中风受伤轴突的单个神经元的鉴定。大部分的标签发生在5层内的轴突和6个神经元在初级感觉皮质覆中风。形象代表中风后7天。比例尺= 500微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53404/53404fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53404/53404fig4.jpg" /> 图4:在中风诱导的使用时间在快速绿色生化和转录化验联合注射白质病变卒中显微切割允许受伤的地区早期识别在24小时（A，上图）。 Immunoblotti纳克特定轴突蛋白可以被执行以表明在笔划单独（A，下图）的区域减少。在较长的时间点，白质中风（左图）的区域可被灶性解剖无特异性标记（B）。右上图显示的去除解剖区域后，白质区域，而右下角的面板显示的包含行程（B）白质解剖区域。 PCR利用RNA从解剖区域从白质（C）中分离特定的少突胶质细胞的基因。 IL =同侧; CL =对侧; C =控制; S =行程。 <table fo:keep-together.within-page="1"><tbody><tr height="21"><td height="21" style="height:21px;"> 注射 </td><td> 前/后 </td><td> 内侧/外侧 </td><td> 背/腹 </td></tr><tr height="21"><td align="right" heigHT ="21"的风格="高度:21px;"&gt; 1 </td><td align="right"> 0.22 </td><td align="right"> 0.22 </td><td align="right"> -2.10 </td></tr><tr height="21"><td align="right" height="21" style="height:21px;"> 2 </td><td align="right"> 0.70 </td><td align="right"> 0.15 </td><td align="right"> -2.16 </td></tr><tr height="21"><td align="right" height="21" style="height:21px;"> 3 </td><td align="right"> 1.21 </td><td align="right"> 0.15 </td><td align="right"> -2.18 </td></tr></tbody></table> 表1:横向成角度的方法（单位mm）立体定向坐标。 <table fo:keep-together.within-page="1"><tbody><tr height="21"><td height="21" style="height:21px;"> 注射 </td><td> 前/后 </td><td> 内侧/外侧 </td><td> 背/腹 </td></tr><tr height="21"><td align="right" heighT ="21"的风格="高度:21px;"&gt; 1 </td><td align="right"> -0.75 </td><td align="right"> -0.96 </td><td align="right"> -2.10 </td></tr><tr height="21"><td align="right" height="21" style="height:21px;"> 2 </td><td align="right"> -1.00 </td><td align="right"> -0.96 </td><td align="right"> -2.05 </td></tr><tr height="21"><td align="right" height="21" style="height:21px;"> 3 </td><td align="right"> -1.25 </td><td align="right"> -0.96 </td><td align="right"> -2.00 </td></tr></tbody></table> 表2:对于后验斜角方法（单位mm）立体定向坐标。

Watch this Scientific Journal Video about A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology at JoVE.com

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

皮层下的多功能小鼠模型白质行程轴索变性和白质神经生物学的研究

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

a-versatile-murine-model-subcortical-white-matter-stroke-for-study

Nanyang Technological University

Research

JoVE Journal

Medicine

8.0K 次观看.  University of California, Los Angeles. Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

视频: A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

小鼠胰岛的分离和包膜下肾移植的方法

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

科研

医学

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

颈动脉支架植入再狭窄的研究的小鼠模型

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

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 ...

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

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

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

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

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 ...

DTI of the Visual Pathway - White Matter Tracts and Cerebral Lesions

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to visual pathways (VP), WMT are not visible with classical MRI or intra-operatively with microscope. DTI will help neurosurgeons to prevent destruction of the VP while removing lesions adjacent to this WMT. We have performed DTI on fifty patients before and after surgery between March 2012 to January 2014. To navigate we used a 3DT1-weighted sequence. Additionally, we performed a T2-weighted and DTI-sequences. The parameters used were, FOV: 200 x 200 mm, slice thickness: 2 mm, and acquisition matrix: 96 x 96 yielding nearly isotropic voxels of 2 x 2 x 2 mm. Axial MRI was carried out using a 32 gradient direction and one b0-image. We used Echo-Planar-Imaging (EPI) and ASSET parallel imaging with an acceleration factor of 2 and b-value of 800 s/mm&#178;. The scanning time was less than 9 min.
The DTI-data obtained were processed using a FDA approved surgical navigation system program which uses a straightforward fiber-tracking approach known as fiber assignment by continuous tracking (FACT). This is based on the propagation of lines between regions of interest (ROI) which is defined by a physician. A maximum angle of 50, FA start value of 0.10 and ADC stop value of 0.20 mm&#178;/s were the parameters used for tractography.
There are some limitations to this technique. The limited acquisition time frame enforces trade-offs in the image quality. Another important point not to be neglected is the brain shift during surgery. As for the latter intra-operative MRI might be helpful. Furthermore the risk of false positive or false negative tracts needs to be taken into account which might compromise the final results.

Diffusion tensor imaging (DTI) was performed to try to depict the major parts of the visual pathway. The goal was to use an FDA approved standard commercial workstation which could be used for everyday routine in order to try to reduce postoperative damage of the visual pathway in the patients.

贸工部视觉通路的 - 白质及脑病变

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

隔离和小鼠主动脉切除;心血管疾病研究的一个多功能的技术

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

一个简单的临界尺寸股骨缺损模型小鼠

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

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 ...

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle screws after spinal surgery, which is a serious concern. To address this problem, diverse types of pedicle screws have been examined to identify those with good fixation strength and osseointegration in spine bone. The porcine spine is a good alternative for the human spine in the evaluation of pedicle screws due to the anatomical size, mechanical characteristics, and cost. Although several studies have reported that pedicle screws are efficient in the porcine model, no study has described detailed protocols for the evaluation of a pedicle screw using the porcine model. Here, we describe a detailed method for evaluating transpedicular screws using an in vivo porcine lumbar spine model. The technical details for anesthesia, spine surgery, and harvest provided here will facilitate with the evaluation of the transpedicular screw fixation model.

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.

用于评估椎弓根螺钉的麻醉，手术和收获方法<em&gt;体内</em&gt;猪腰椎模型

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle ...