作者感谢丹Firl，克里斯·科贝特和杰西·丹森博士。资金是SR提供美国国立卫生研究院NINDS R01 NS060765，在P30铜业试点方案，LJ和儿童健康签名计划，以LJ在新墨西哥大学。

机构护理和使用委员会在两个波士顿儿童医院和新墨西哥健康科学中心大学批准的所有实验程序。 注:在开始的过程中，密封，消毒和高压灭菌所有的手术器械和手术单。此外，准备在无菌小瓶术后药物，包括0.125％bipivucaine和0.1mg / kg的丁丙诺啡。还制备脂多糖（LPS）溶液无菌:0.04毫克/毫升的LPS（0111:B4）在含有稀释伊文思蓝染料的无菌盐水。 1.麻醉<ol><li>诱导麻醉在胚胎第18天（E18）孕只SD大鼠用3％异氟烷平衡70％氮气和30％氧气的混合物。 </li><li>从感应腔室除去大鼠和放置在披外科循环水毯设定在37℃的鼠仰卧。转移麻醉鼻锥和减少isofluraね级至2％。 </li><li>轻轻涂抹眼药膏每只眼睛防止角膜干燥。在手术过程中连续监控温度，呼吸率和动物的心脏费率。产妇生理应在整个过程保持稳定。 </li></ol> 2.手术准备和磨砂<ol><li>使用小动物快船去除所有毛发的下腹部区域。剃须的矩形图案小心，以避免切口乳头或生成剃刀皮疹，可刺激活出生的幼仔未来护理。 </li><li>通过交替应用的聚维酮碘和70％乙醇擦洗，用无菌棉签准备腹部皮肤。重复擦洗，这样碘伏和70％乙醇分别适用3倍以交替的方式。让我们干。 </li><li>通过没有脚趾捏反射确认麻醉深度。在不存在反射和刺激的疼痛，减少异氟烷级到1％。 </li><li>使用无菌手术巾，悬垂的动物。照顾使得它们最大化冲洗流体它们吸收，同时不妨碍血液流动到子宫角的量放置窗帘以适当角度。 </li></ol> 3.腹部剖腹<ol><li>用手术刀做一个3厘米中线切口中制备的腹部皮肤。说白了解剖，从腹部筋膜，用剪刀将皮肤层。使用镊子和手术剪，提升腹部筋膜层和使无血管白线的切口来访问腹膜腔。 </li><li>放置外科纱布上切口的外观和滋润用无菌盐水。使用钝钳和外部的压力放在腹部，轻轻地从腹腔内取出子宫角，并安排在湿润的纱布。 </li><li>小心避免纠缠和肠道接触。使用镊子胎儿在羊水个人囊之间仅接触肌肉组织安排。揭露和使用钝性分离分离4子宫动脉。 注:必须小心解剖子宫动脉。周围组织和血管本身是极其微妙的。损坏产妇的血管可引起出血，严重的情况下，胎儿和产妇死亡。 </li></ol> 4.放置动脉瘤夹中<ol><li>将每个子宫动脉老鼠地下30动脉瘤夹。确保停止血液流动，包括近端的子宫血管包括个人胎盘和远端脉冲，并且变黑。覆盖暴露的角，整个手术视野用纱布和灌溉用无菌生理盐水。请注意，以保持田间湿润灌溉大约每隔10分钟。 </li><li> 60分钟后，取出纱布和灌溉领域。确保子宫角和船只被充分润湿成功的夹子拆除。轻轻地使用镊子删除每个动脉瘤夹。注意不要造成创伤的船只，并MAINT拆卸过程中，AIN组织的完整性。 </li><li>彻底浇子宫角和领域，同时注意从羊膜囊取出纱布任何杂散线程。 </li></ol> 5.注射脂多糖到羊膜囊<ol><li>在每个单独的羊膜囊的底部，只是前胎盘板，注入100μl的LPS（4微克/囊）与稀释伊文思蓝染料的羊水。使用钝钳稳定和中用于注射的最佳位置旋转每个羊膜囊。稀埃文的蓝色染料是一种造影剂在确认正确放置的注射器和注射有帮助。 注:只能使用超细0.3毫升胰岛素注射器连接8毫米一个31 G针的羊膜腔内注射。使用较大的表针会导致慢性羊水流失，胎儿死亡和怀孕的重吸收。在去除注射器少量羊水泄漏的可mitig通过直接压到羊膜囊ated。有些老鼠胎儿可能会容忍一定程度羊水过少。然而，从穿刺急性羊水流失大表针，或导致慢性液体泄漏意外爆胎，导致胎儿流产，并在严重的情况下，相邻的怀孕损失。 </li><li>灌溉子宫角3倍用无菌盐水。 </li></ol> 6.关闭剖腹<ol><li>使用镊子，小心地返回子宫角，以腹腔。确保羊膜囊和中线切口之间有足够的空间，再利用近似正在运行的3-0丝线缝合肌筋膜层的边缘。请注意放置羊膜囊的同时关闭肌肉切口。注意不要缝合进入或通过一个囊。 </li><li>重新近似皮肤层，利用一个运行3-0丝线缝合，闭合皮肤层。 注:剖腹应在连续的缝合线的两层，以允许被关闭对皮肤和肌肉的扩张而增加妊娠。连续缝合允许均匀分布的伤口张力。较少所需间断缝合作为多结是刺激性，可以很容易地通过鼠在从麻醉中恢复咀嚼。手术缝钉被不希望的。手术结尾巴应该剪得很短（&lt;3毫米）。 </li><li>注射1毫升使用＆G针0.125％布比卡因周围皮下伤口边缘。施用一种剂量为0.1mg / kg的丁丙诺啡皮下的颈背。 </li><li>关闭必要异氟醚并用毛巾擦干老鼠。放置在干净的笼子和监测从麻醉中恢复。确保鼠不会成为低温。 </li></ol> 7，术后恢复和护理<ol><li>监视老鼠每8-12小时为72小时，然后每天，直到幼崽出生（约E22或E23）。辖附加剂量的丁丙诺啡q8-12小时/ 72小时或PRN如由IACUC。 </li><li>监测大鼠疼痛，不适，阴道出血或从手术部位出血的迹象。检查缝线和切开和照顾，以确保老鼠不会咀嚼或删除其缝线过早。尽管非常罕见，老鼠危及他们的缝线有可能对伤口裂开。 注:有时只可摄取的床上用品和非食品的过量，称为异食癖，丁丙诺啡管理的副作用。虽然很罕见，大鼠必须为异食癖和潜在的后续肠梗阻进行监控。 </li></ol> 8.组织处理和Cryosectioning <ol><li>为了准备苏木精 - 伊红（H＆E）在出生后大脑的染色，除去从他们的家笼幼崽在出生后第2天（P2）。用手术剪斩首幼鼠，轻轻地从头骨取出大脑。 </li><li>滴修复脑在15ml锥形管中含7 ml的4％多聚甲醛的磷酸盐缓冲盐水（PBS）中。将大脑在4℃和修复72小时。 </li><li> 72小时后，转移到大脑含有30％蔗糖的无菌PBS溶液（重量/体积），并返回到4℃。 注意:一旦大脑下降的蔗糖溶液中，他们准备在低温恒温器进行切片。 </li><li>快速冷冻大脑和安装在低温恒温器杵收购冷冻冠状切片。切20微米的冰冻冠状切片并安装在载玻片上。确保部分被连续收集。 </li><li>允许滑动到在室温下干燥过夜。商店滑动于-20℃。 </li></ol> 9.苏木精 - 伊红染色<ol><li>以滑动安装的冰冻切片和温至室温。 注:准备所有的解决方案新鲜。 </li><li>地方上滑动一个2小时滑动温暖设定为50℃。 </li><li>转移到幻灯片染色架。浸滑10X双蒸去离子水（DDH 2 O的）。 </li><li>孵育在100％的苏木玻片5分钟。钛我在苏木可以根据紫色染色的程度进行优化。 </li><li>浸幻灯片4倍的自来水H 2 O和静置在干净的自来水H 2 O的1分钟。 </li><li>浸滑动15倍酸性醇（250毫升70％乙醇+ 1毫升浓盐酸）。 </li><li>浸幻灯片4倍的自来水H 2 O和静置在干净的自来水H 2 O的1分钟。 </li><li>孵育在1％碳酸锂2分钟。 </li><li>浸幻灯片4倍的自来水H 2 O和静置在干净的自来水H 2 O的1分钟。 </li><li>孵育在95％乙醇1分钟。 </li><li>浸幻灯片7倍，100％曙红。在曙红骤降的数量可以根据粉色染色程度进行修改。 </li><li>浸载玻片5倍的95％乙醇。 </li><li>浸滑5倍的95％乙醇的新鲜变化。 </li><li>孵育在100％乙醇载玻片1分钟。 </li><li>在100％乙醇1分钟新鲜变化孵育载玻片。 </li><li>孵育在100％二甲苯幻灯片15分钟。 注:二甲苯步骤和盖片应该发生在通风橱中。 </li><li>孵育在二甲苯中15分钟新鲜变化幻灯片。 </li><li>盖玻片Permount，让干燥通风橱。 </li><li>图片幻灯片用光学显微镜。 </li></ol>

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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=Arrested+oligodendrocyte+lineage+maturation+in+chronic+perinatal+white+matter+injury.">Arrested oligodendrocyte lineage maturation in chronic perinatal white matter injury.</a> Ann Neurol. 63, 520-530 (2008).</li><li>Robinson, S., Li, Q., Dechant, A., Cohen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+loss+of+gamma+aminobutyric+acid+pathway+expression+after+human+perinatal+brain+injury.">Neonatal loss of gamma aminobutyric acid pathway expression after human perinatal brain injury.</a> J Neurosurg. 104, 396-408 (2006).</li><li>Boksa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+prenatal+infection+on+brain+development+and+behavior+a+review+of+findings+from+animal+models.">Effects of prenatal infection on brain development and behavior a review of findings from animal models.</a> Brain Behav Immun. 24, 881-897 (2010).</li><li>Burd, I., Brown, A., Gonzalez, J. M., Chai, J., Elovitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+term+chorioamnionitis+unraveling+causes+of+adverse+neurological+outcomes.">A mouse model of term chorioamnionitis unraveling causes of adverse neurological outcomes.</a> Repro Sci. 18, 900-907 (2011).</li><li>Bergeron, J. 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作者什么都没有透露。

早产儿脑病是困难的，因为病因，神经发育时间的推移，人类的大脑网络形成的复杂性，重叠损伤机制的复杂的相互作用的动物模型，以及中枢神经系统的不同表​​型侮辱体现在人类的早产儿。期末与特定细胞类型的漏洞相关联（即未成熟的少突胶质细胞）21，以及不同的发育调节的途径（即底板，膜转运和受体亚单位）12,13,22。然而，当动物模型复制人类生存条件尽可能地可显著的进步。在这里，我们开发了一个模型产前侮辱结合的中枢神经系统损伤的早产儿观察机制的异质性，从而为灰质和白质损伤和恢复的后续评估。在人类中，升细菌感染削弱羊膜和precipita膜忒早破。此外，胎盘灌注缺损强调胎盘接口和破坏胎盘的动态平衡。因此，胎盘underperfusion从宫内感染化合物中枢神经系统损伤。无可否认，它是具有挑战性的模拟上行它之前绒毛膜在啮齿类动物，因为它们有一个双工子宫细菌感染的常见临床情况。每个子宫角都有自己的宫颈，以及多胎妊娠的同时进行。尽管存在这些挑战，临床前模型已适应涉及母体胎盘的胎儿单元的多个组件和在子宫内炎症掺入到不同程度。而没有个体临床前模型中是理想的，以测试每个特定假说，本文所描述的模型结合的细胞和分子异常，行为和功能障碍，母体胎盘的胎儿系统，以及宫内感染和共同到如此毫安胎盘炎症成分纽约早产20,23。 种用于选择建模期末影响的实验数据的解释中，由物种造成的固有限制的范围内。简单的说，生不等于在所有动物24相似点中枢神经系统的发展。这里描述的模型可以同时在孕小鼠和大鼠进行，但幼崽的生存小鼠是显著的经验或强调水坝下降。符合我们之前的报道，出生时（P0）大鼠胎儿流产是TSHI + LPS动物（约40％）相比，假，LPS和TSHI单独增加，但幸存的幼崽不通过P28 20表现出显著的重量差异。类似物种之间的差异，损伤的妊娠期间的定时在后代的神经发育轨迹了至关重要的作用。增殖，迁移和迪菲的神经细胞发育阶段的时空调控rentiation之间区别各种哺乳动物24-26。这些细胞特异性的发育程序影响易受伤害。例如，少突胶质细胞谱系和GABA能神经元发育的早产的定时的定时的重叠使得这些细胞特别容易围产期辱骂27-29。因此，这种模式在E18大鼠开发（并成功地转换为E17小鼠在C57BL / 6背景），因为这正对应于发生在人类婴儿之前，极早产在23-25 ​​周的妊娠20宫内全球产前侮辱。我们以前表明O4免疫反应未成熟的少突胶质细胞受到的影响最大，在这个发展阶段16。其减轻，结果表明具有降低的存活和成熟14，具有最显着的减少O4 +和谱系16，与先前的报道一致的O1 +阶段由其他研究者30。此外，我们已经证明过早脱落辅助板，减少了KCC2的表达，降低癫痫发作阈值和受损的步态12,13,15失调的GABA能信号的早产儿31是一致的。 这里所描述的模型提供较以前的型号中用于研究的早产23围产期脑损伤的啮齿动物许多好处。它采用了全产妇胎盘，胎儿系统并导致大脑和胎盘损伤。之前我们已经发布的假之间的比较，TSHI独自一人，LPS单独TSHI + LPS和分级TSHI 16在功能性成果和生化20，和不同的差异。虽然单侧颈动脉结扎和新生大鼠全身缺氧的事先调查已摆脱机械洞察无数的病理生理过程（ 即未成熟的少突胶质细胞对缺血的敏感性），直接转化和临床relevaNCE这种模式是不太可靠的。除了所描述的应用中，这里所描述的模型可以是一个信息的工具，由早产影响其他器官系统，包括坏死性小肠结肠炎（NEC），心脏，肺，肾和下丘脑 - 垂体轴功能障碍调查。由于脂多糖药理学的复杂性和在母亲和胎儿药效差异，腹膜内注射LPS在水坝的可能性较小，以产生此处所示的相同胎儿炎症反应。此外，内毒素不会穿过胎盘可靠20,32。以前，我们试图直接宫颈应用LPS和宫腔注射类似于在其他小鼠模型33被描述。然而，我们发现，死亡率CNS损伤的和不一致，显著幼仔间同窝内增加。这里，4微克/囊的剂量使用剂量 - 反应实验进行优化。增加剂量的LPS给予AMNiotic仓导致增加胎儿的死亡率。脂多糖具有优于直接感染典型宫内革兰氏阴性细菌，它通过激活通过toll样受体4炎症信号，而不会导致活性的细菌感染和病原体传播的相关风险。然而，这种模式可被修改成包括自人胎盘，包括B组链球菌 ，这会导致胎盘和神经病理学异常，并在大鼠34孤独症样行为分离常见病原体和微生物。同样，解脂蛋白多条带状抗原可以模拟解的物种感染。由于解是人类绒毛膜羊膜炎35的最常见的原因，这也可能是一个渠道为未来的调查。随着越来越多的灭活感染剂变得可用时，将信息，以确定他们的差异如何影响神经发育和神经修复干预的功效。这种模式的局限性包括从羊膜腔内注射羊水流失。虽然没有影响注意用无菌盐水羊膜腔内注射，用于执行注射针的型号是一个关键的技术元素。必须注意不要用针大于31 G.手术并发症的母鼠相关的剖腹手术是极为罕见的，包括伤口裂开，肠梗阻，腹膜炎和怀孕的完全丧失，孕产妇死亡率低于5％。

随着37周之前估计胎龄1出生在美国的婴儿超过12％，围产期脑损伤（PBI）的早产儿是永久性残疾的显著原因。 PBI从早产，早产儿也称为脑病（EOP），影响整个中枢神经系统（CNS）。 CNS损伤往往开始在子宫内，并且由产前过程，包括绒毛膜和产后并发症如低氧和败血症加剧。 PBI从全身辱骂改变神经发育，导致脑性麻痹，癫痫，认知延迟和众多的神经精神障碍影响情绪调节，记忆和执行功能1,2-。虽然已经取得了很大的进步，在有限的理解仍然是来自早产中枢神经系统损伤的细胞和分子的后果如何转化到谁出生的早产儿在众人的神经系统后遗症。这种缺乏知识后的中枢神经系统损伤严重程度ERS实时诊断和新兴干预措施的通知剂量。此外，对于这些弱势患者群体年龄适当的治疗策略仍然难以实现。 宫内炎症是极度早产非常普遍，涉及复杂的胎儿产妇胎盘炎症级联3。宫内感染通常是亚临床型。具体胎盘发现与急性炎症，或组织学绒毛膜羊膜炎一致的，都是胎儿炎症反应的主要决定因素和重合与早产3-5有关的脑损伤。事实上，胎儿的炎症反应有从早产长期预后不同的临床意义。婴儿谁是小于胎龄儿（SGA）或谁遇到感染是非常脆弱的神经功能障碍3,4。绒毛膜羊膜炎是一种典型的病理诊断以下早产<sup&gt; 6,7，和组织学检查发现炎症在胎盘中70％的出生非常早产4例迹象。此外，绒毛膜羊膜炎与认知功能障碍为两年8有关。在出生非常早产儿的产妇胎盘血管underperfusion的证据也与脑瘫儿童9关联。的绒毛膜羊膜炎及胎盘灌注缺损的协同影响在两年10,11岁是很好地说明通过非正常神经成果在该患者人群中显着高风险。 以模仿与病原体诱导的炎症相关的人类全身胎盘灌注缺损和绒毛膜，我们开发产前瞬态全身缺氧缺血（TSHI）结合羊膜腔脂多糖（LPS）的大鼠模型。我们的目标是要调整我们的TSHI模型独自在大鼠12-16包括宫内炎症，以方便与早产有关的中枢神经系统损伤的临床前模型。 TSHI单独揭示少突胶质细胞谱系细胞，皮层神经元的持续损失，增加的细胞死亡，和升高的促炎细胞因子水平，以渐进的缺血间隔导致伤害与产前脑损伤16一致的分级图案。修改该模型的缺血性成分也显示在记忆编码，短期和长期记忆和轻度肌肉骨骼改变障碍研究，因为它们17-19的年龄。实际上，我们以前表明，TSHI + LPS的组合概括期末的病理生理特点，包括少突胶质细胞和神经元损失，轴突损伤，细胞炎症和功能异常20。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Saline Solution, 0.9%</td>
			<td >Sigma</td>
			<td >S8776</td>
			<td ></td>
		</tr>
		<tr >
			<td >LPS 011B4</td>
			<td>Sigma</td>
			<td>L2630</td>
			<td></td>
		</tr>
		<tr >
			<td >Evan&#39;s Blue Dye</td>
			<td>Sigma</td>
			<td>E2129</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical gloves</td>
			<td>Biogel</td>
			<td>40870</td>
			<td></td>
		</tr>
		<tr >
			<td >OR Towels</td>
			<td>Cardinal Health</td>
			<td>287000-008</td>
			<td>Sterile</td>
		</tr>
		<tr >
			<td >PDI Alcohol Prep Pads</td>
			<td>Fisherbrand</td>
			<td>06-669-62</td>
			<td></td>
		</tr>
		<tr >
			<td >Mini Arco Rechargeable Clippers</td>
			<td>Kent Scientific Corp.</td>
			<td>CL8787</td>
			<td></td>
		</tr>
		<tr >
			<td >Betadine surgical scrub</td>
			<td>Purdue Products L.P.</td>
			<td>67618-151-17</td>
			<td></td>
		</tr>
		<tr >
			<td >Eye Lubricant</td>
			<td>Refresh Lacri Lube</td>
			<td>00023-0312</td>
			<td></td>
		</tr>
		<tr >
			<td >Blunt Forceps</td>
			<td>Roboz</td>
			<td>RS-8100</td>
			<td></td>
		</tr>
		<tr >
			<td >Scissors</td>
			<td>Roboz</td>
			<td>RS-6808</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical Scissors</td>
			<td>Roboz</td>
			<td>RS-5880</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical Scissors</td>
			<td>F.S.T.</td>
			<td>14002-16</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td>1 ml</td>
		</tr>
		<tr >
			<td >Needle</td>
			<td>BD</td>
			<td>305122</td>
			<td>25G 5/8</td>
		</tr>
		<tr >
			<td >Needle</td>
			<td>BD</td>
			<td>305128</td>
			<td>30G 1</td>
		</tr>
		<tr >
			<td >Cotton-tipped Applicators</td>
			<td>Fisherbrand</td>
			<td>23-400-114</td>
			<td>Small, 6 inch sterile</td>
		</tr>
		<tr >
			<td >Cotton Gauze Sponge</td>
			<td>Fisherbrand</td>
			<td>22-362-178</td>
			<td></td>
		</tr>
		<tr >
			<td >Needle Holders</td>
			<td>Kent Scientific Corp.</td>
			<td>INS600109</td>
			<td>12.5 CM STR</td>
		</tr>
		<tr >
			<td >Vessel Clips</td>
			<td>Kent Scientific Corp.</td>
			<td>INS600120</td>
			<td>30G Pressure</td>
		</tr>
		<tr >
			<td >3-0 Perma Hand Silk Sutures</td>
			<td>Ethicon</td>
			<td>1684G</td>
			<td>Black braided, 3-0 (2 metric), 18&#34;, non-absorbable,&#160; PS-1 24mm needle, 3/8 circle</td>
		</tr>
		<tr >
			<td >Insulin Syringes</td>
			<td>BD</td>
			<td>328438</td>
			<td>0.3cc 3mm 31G</td>
		</tr>
		<tr >
			<td >Pentobarbital</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Buprenorphine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Bupivacaine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Lithium Carbonate</td>
			<td>Acros Chemicals</td>
			<td>554-13-2</td>
			<td></td>
		</tr>
		<tr >
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			<td>48311-703</td>
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modeling encephalopathy of prematurity using prenatal hypoxia-ischemia with intra-amniotic lipopolysaccharide in rats

早产儿的脑病（EOP）是包括中枢神经系统（CNS）的异常与早产有关的术语。为了最好预先平移目标，并发现新的治疗策略用于与早产有关的脑损伤，期末的临床前模型必须包括类似的机制产前全球损伤的观察人类和涉及的产妇胎盘，胎儿系统的多个组件。理想的情况下，模型应该产生的功能缺损的类似光谱在成熟动物和概括的病理生理的多个方面。为了模仿人体全身胎盘灌注缺损，胎盘underperfusion和/或绒毛膜羊膜炎与早期早产病原体引起的炎症有关，我们开发了产前瞬时全身缺血缺氧（TSHI）联合羊膜腔内脂多糖（LPS）的模型。在怀孕的SD大鼠，TSHI通过子宫动脉栓塞Øñ胚胎18天（E18）诱导随着胎儿中枢神经系统损伤有关分级胎盘underperfusion缺陷。当羊膜腔内注射LPS结合，胎盘炎症增加，中枢神经系统损伤中配合相关的白质，步态和影像学异常。产前TSHI和TSHI + LPS产前侮辱满足几个的期末模型，包括扼要宫内侮辱的标准，造成神经元，少突胶质细胞，板式损失和功能障碍在模仿那些在极其出生的孩子发现成年动物的损失早产。此外，这种模式使得炎症引起的不同损伤类型的清扫。

以下TSHI + LPS在E18，苏木精和曙红染色显示在两个胎盘显著组织病理学异常（ 图1）和在脑中（图2）。胎盘检查了E19和E21都严重水肿与微出血，坏死整个蜕膜和迷宫。显著炎性浸润和血管分布增加，也观察到。脑检查了P2显示脑室扩大，以及白质和底板神经元的损失相比，沙姆斯。此前，我们曾报道了TSHI + LPS诱导炎症反应，并产生持久的白质和青壮年20伴随显著运动障碍性轴索异常。 TSHI + LPS也显著降低氯化钾协同转运2（KCC2）蛋白表达，氯化物协同转运至中央的γ氨基丁酸的发展（GABA）的GABA能抑制，在皮层在P15（图3 </STRONG&gt;，* P &lt;0.05）。这些数据与KCC2的发育障碍一致的上调在电路成熟的关键产后和这些调查结果与我们之前的TSHI的效果独自在海马CA3区13报道一致。 <img alt="图1" src="/files/ftp_upload/53196/53196fig1.jpg" /> 图1:TSHI + LPS诱导显著组织学异常胎盘短暂性的宫内缺血缺氧和羊膜腔内注射LPS对胚胎18天（E18），从E19 TSHI + LPS胎儿（B）胎盘是严重水肿伴出血（箭头），坏死，增加炎症浸润比较，假手术（A，比例尺= 100微米）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53196/53196fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图2:TSHI + LPS诱导显著组织学异常的大脑短暂性的宫内缺血缺氧和羊膜腔内注射LPS对胚胎18天（E18），出生后脑室，底板神经元和白质损失的幼崽进行观察双TSHI + LPS（B）相比，假手术（A）敏锐地在P2。 （比例尺= 100微米; SP =底板; WM =白质; LV =侧脑室） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53196/53196fig2large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53196/53196fig3.jpg" /> 图3:TSHI + LPS减少KCC2表达从存储进行Western印迹。微解剖皮层组织，显示在宫内瞬时全身缺血缺氧和羊膜腔内注射LPS对胚胎18天brane制剂（E18）显著降低KCC2，神经元特异性氯化钾共转运中心表达发展集成脑电路和抑制，在15日龄（N = 6-10，平均值±SEM，斯氏t检验，* p &lt;0.05）。

Watch this Scientific Journal Video about Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats at JoVE.com

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

早产儿脑病建模使用产前缺氧缺血大鼠羊膜腔内毒素

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

modeling-encephalopathy-prematurity-using-prenatal-hypoxia-ischemia

Nanyang Technological University

Research

JoVE Journal

Medicine

11.2K 次观看.  University of New Mexico. Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

视频: Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

建模在小鼠体内的行程 - 中东与灯丝模型大脑中动脉闭塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

科研

医学

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

利用双光子显微镜在慕尼黑Wistar大鼠量化肾小球通透性荧光大分子

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

无创成像和脑缺血分析生活中的大鼠使用正电子发射断层扫描<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

大鼠使用超声技术药物引起的肾血流动力学障碍的早期检测

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

新生儿缺氧缺血性脑病的小猪模型

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

评估传染性海绵状脑病物种界限与<em&gt;在体外</em&gt;朊蛋白转化测定法

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

同时PET / MRI成像在小鼠脑缺氧缺血

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

基于短暂性脑缺氧缺血性血栓性中风模型

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

外切囊梗死模型使用光化学技术

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.