Name: modeling encephalopathy of prematurity using prenatal hypoxia-ischemia with intra-amniotic lipopolysaccharide in rats
Uploaded: 2015-11-20T14:00:00.000+00:00
Duration: 7 min 36 s
Description: Watch this Scientific Journal Video about Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats at JoVE.com

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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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><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&#039;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&quot;, non-absorbable,&amp;nbsp; PS-1 24 mm needle, 3/8 circle</td></tr><tr><td>Insulin Syringes</td><td>BD</td><td>328438</td><td>0.3 cc 3 mm 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><td>Superfrost Plus Microscope Slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Hematoxylin</td><td>Leica</td><td>3801521</td><td>Surgipath Gill II Hematoxylin</td></tr><tr><td>Eosin</td><td>Leica</td><td>3801601</td><td>Surgipath Eosin</td></tr><tr><td>Xylenes</td><td>Fisherbrand</td><td>X3S-4</td><td>Histological Grade</td></tr><tr><td>Permount</td><td>Fisherbrand</td><td>SP15-100</td><td></td></tr><tr><td>Coverglass</td><td>Fisherbrand</td><td>12-548-5P</td><td>Fisher Finest Premium Coverglass</td></tr></tbody></table>

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/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/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

Universidad San Sebastian

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

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

脑室周围白质软化的小鼠模型

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

科研

神经科学

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

围产期缺血缺氧缺血性脑病模型

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

生物学

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 ...

医学

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

小鼠新生儿缺氧缺血性脑损伤模型的神经行为评估

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

轻度宫内灌注圈狭窄的大鼠模型

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

A Ferret Model of Inflammation-sensitized Late Preterm Hypoxic-ischemic Brain Injury

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for infants with the neurological sequela of prematurity. Ferrets are ideal candidates for modeling the preterm human brain, as they are born lissencephalic and develop gyrencephalic brains postnatally. At birth, ferret brain development is similar to a 13 week human fetus, with postnatal-day (P) 17 kits considered to be equivalent to an infant at 32&#8211;36 weeks&#39; gestation. We describe an injury model in the P17 ferret, where lipopolysaccharide administration is followed by bilateral cerebral ischemia, hypoxia, and hyperoxia. This simulates the complex interaction of prolonged inflammation, ischemia, hypoxia, and oxidative stress experienced in a number of neonates who develop brain injury. Injured animals display a range of gross injury severity, with morphological changes in the brain including narrowing of multiple cortical gyri and associated sulci. Injured animals also show slowed reflex development, slower and more variable speed of locomotion in an automated catwalk, and decreased exploration in an open field. This model provides a platform in which to test putative therapies for infants with neonatal encephalopathy associated with inflammation and HI, study mechanisms of injury that affect cortical development, and investigate pathways that provide resilience in unaffected animals.

The method describes inflammation-sensitized hypoxic-ischemic and hyperoxic brain injury in the P17 ferret to model the complex interaction between prolonged inflammation and oxidative brain injury experienced in a number of late preterm infants.

炎症敏感晚期缺氧缺血性脑损伤的Ferret模型

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for ...

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and lifelong neurological deficits. While PHH can be treated by temporary and permanent cerebrospinal fluid (CSF) diversion procedures (ventricular reservoir and ventriculoperitoneal shunt, respectively), there are no pharmacological strategies to prevent or treat IVH-induced brain injury and hydrocephalus. Animal models are needed to better understand the pathophysiology of IVH and test pharmacological treatments. While there are existing models of neonatal IVH, those that reliably result in hydrocephalus are often limited by the necessity for large-volume injections, which may complicate modeling of the pathology or introduce variability in the clinical phenotype observed.
Recent clinical studies have implicated hemoglobin and ferritin in causing ventricular enlargement after IVH. Here, we develop a straightforward animal model that mimics the clinical phenotype of PHH utilizing small-volume intraventricular injections of the blood breakdown product hemoglobin. In addition to reliably inducing ventricular enlargement and hydrocephalus, this model results in white matter injury, inflammation, and immune cell infiltration in periventricular and white matter regions. This paper describes this clinically relevant, simple method for modeling IVH-PHH in neonatal rats using intraventricular injection and presents methods for quantifying ventricle size post injection.

We present a model of neonatal intraventricular hemorrhage using rat pups that mimics the pathology seen in humans.

通过脑室内注射血红蛋白模拟新生儿脑室内出血

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term ...

Modeling Posthemorrhagic Hydrocephalus of Prematurity in Rats

Posthemorrhagic hydrocephalus of prematurity (PHHP) is a serious sequela of severe intraventricular hemorrhage (IVH) in very preterm infants less than 32 weeks gestational age (GA). PHHP is defined by the accumulation of cerebrospinal fluid (CSF) associated with clinical symptoms of elevated intracranial pressure (ICP). Infants with PHHP suffer lifelong shunt dependence, with half requiring repeat surgery in the first year of life and many requiring multiple additional surgeries throughout the lifespan. Prenatal chorioamnionitis predisposes preterm infants to severe IVH and the need for surgical treatment of PHHP trends with neonatal sepsis. These clinical features suggest that systemic inflammation is an integral component of PHHP pathophysiology. 
Here, we define an animal model that recapitulates all clinical aspects and essential features of PHHP in rats. The goal of this protocol is to illustrate how in utero chorioamnionitis and postnatal IVH using lysed red blood cells can be combined to yield PHHP. This preclinical approach yields progressive macrocephaly and domed craniums, elevated intracranial pressure, and ventriculomegaly that can be detected via magnetic resonance imaging (MRI) or via microscopy. In addition to sustained disruption in CSF dynamics, rats also have cognitive delay and functional disability into adulthood. Accordingly, this preclinical platform facilitates unique and unparalleled translational studies of PHHP that can incorporate molecular, cellular, biochemical, histologic, imaging, and functional outcome measures. It can also be used for rigorous analysis of the choroid plexus, ependymal motile cilia, and glymphatic system in parallel. Last, it can also be an invaluable preclinical tool for the investigation of novel surgical intervention strategies and non-surgical therapeutic approaches for the treatment of hydrocephalus.

Posthemorrhagic hydrocephalus of prematurity (PHHP) can be modeled in neonatal rats by combining chorioamnionitis and intraventricular hemorrhage. The combination of these prenatal and postnatal events accurately recapitulates the clinical hallmarks of PHHP, including macrocephaly, ventriculomegaly, and elevated intracranial pressure, through the lifespan.

大鼠早产儿出血后脑积水建模

Posthemorrhagic hydrocephalus of prematurity (PHHP) is a serious sequela of severe intraventricular hemorrhage (IVH) in very preterm infants less than 32 ...

Induction of Cerebral Microhemorrhages in a Rat Model Using Lipopolysaccharide

Source: Li, D., et al. <a href="https://app.jove.com/v/58423">Sub-acute Cerebral Microhemorrhages Induced by Lipopolysaccharide Injection in Rats.</a> J. Vis. Exp. (2018).
This video demonstrates the process of inducing blood-brain barrier (BBB) disruption and cerebral microhemorrhages in a rat model using lipopolysaccharide (LPS). It highlights the sequence of immune responses that lead to increased BBB permeability and subsequent brain tissue damage.

使用脂多糖诱导大鼠模型脑微出血

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...