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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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. 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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?...

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。

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			<td>Roboz</td>
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			<td>Roboz</td>
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			<td>F.S.T.</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?...

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 ...

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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.