Name: induction of maternal immune activation in mice at mid-gestation stage with viral mimic poly(i:c)
Uploaded: 2016-03-25T13:00:00.000+00:00
Duration: 7 min 13 s
Description: Watch this Scientific Journal Video about Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C at JoVE.com

我们要纪念已故保罗·帕特森博士对MIA模型，自闭症和精神分裂症研究的进步作出贡献。我们承认萨尔基斯K. Mazmanian就这一协议的大力支持;鲁本·M.巴戎寺，伊薇特·加西亚 - 弗洛雷斯，卡伦C.伦乔尼，和Leslie A.诺伊曼行政援助;阿里Khoshnan和Jan C.柯对拍摄的援助;伊莱恩Y.萧和Natalia Malkova它们对MIA感应意见;杰弗里·科克伦S.，华金·古铁雷斯，关F·李，海梅·罗德里格斯，洛雷娜C.桑多瓦尔，和Natalie A.贝尔杜斯科及专家畜牧业。这项工作是由美国国立卫生研究院康特中心奖（NIH 5P50MH086383-04，保罗·H·帕特森）的支持;自闭症（＃7670，保罗·H·帕特森）;西蒙斯基金会（＃322839，以萨尔基斯K. Mazmanian）;美国国立卫生研究院培训资助（NIH / NRSA T32GM07616到K-HC）;加州理工大学暑期大学生研究奖学金（SURF）（以ZY）;安进学者计划在加州理工学院（ZY到）;和博士后奖学金FROM国家科学委员会，台湾（NSC 101-2917-I-564-039，到W-LW）。

所有协议均技术机构动物护理和使用委员会的加州理工学院（IACUC）的批准下进行的。 1.准备定时交配双<ol><li>使用基因表达分析至少6个水坝的行为实验和3。 注意:使用估计雌性小鼠的双数，如只有50％左右的小鼠将从定时交配堵塞。 </li><li>使用雌性小鼠事先没有怀孕，并在8-16周龄。 注:雌性小鼠有性行为之前接触到雄性小鼠但一直没怀孕是可以接受的。 </li><li>房子的雌性小鼠在一起并放置笼彼此相邻，以同步它们的动情周期。使用标准的小鼠笼的超过550英寸的高度和一个75平方英寸的室内地板，以允许5成年小鼠的外壳。标签使用耳朵打孔每个雌性小鼠。 </li></ol> 2.设置定时垫ING对<ol><li>在暗循环开始前设置定时交配0-2小时。将两个雌性小鼠每个笼子。取出老鼠并单独衡量他们。记录体重每雌性小鼠。 </li><li>放置一个雄性小鼠与两个雌性小鼠每笼。使用三人配合以最小化父系混杂影响。 </li></ol> 3.阴道塞检查（E0.5） <ol><li>检查雌性小鼠0-4小时黑暗循环结束后。轻轻抬起雌性小鼠从尾的基部，并允许小鼠抓住线栅，笼顶部，或笼边。 </li><li>搜索阴道塞的迹象:阴道塞在阴道口可见白色肿块。如果插头并不明显，轻轻插入200微升的枪头插入阴道口。从抗凝血证实阴道栓形成。检查阴道塞每天一次。 注:阴道塞是只指示已发生交配，因此不克uarantee妊娠。阴道塞可能难以在小鼠中的某些菌株鉴别。求助有经验的护理人员小鼠增加的插件识别的准确性。 </li><li>移动插入小鼠到一个新的笼子和组安置他们。定义阴道塞的外观胚胎0.5天（E0.5）的日子。 </li></ol> 4.在E10.5-11.5，检查是否怀孕<ol><li>轻轻提起尾巴的基部，并允许小鼠抓住线栅，笼顶部，或笼边。观察腹部区域以检查怀孕， 例如，在腹部的凸起（ 图1）的迹象。 注意:怀孕的迹象总是在E11.5比E10.5更加明显。 </li><li>称量小鼠来验证怀孕。孕小鼠通常重达在E10.5重约20％，并在E12.5重30％相比，非妊娠小鼠（ 图2）。单家在清洁笼子里的孕鼠。 </li></ol> 5. 20毫克/公斤的聚（I:C）的制备<ol><li>使用每组至少6水坝进行行为实验和3元组基因表达分析。制备多聚（I:C）的相应数量的解决方案。 </li><li>称量该聚（I:C）的粉末在50ml的锥形管，使40毫克/毫升的最终浓度。为了最小化和标准化引起的喷射量的应力，注入5微升的聚（I:C）对小鼠体重（5微升/克，或5毫升/公斤）每克溶液。为了诱导MIA，我们注入20毫克聚:每公斤（I C）。 注:聚的浓度（I:C）所需的解决方案是（20毫克/千克）/（5毫升/公斤）= 4毫克/毫升。由于该聚（I:C）的粉末含有10％的聚（I:C），我们需要使（4毫克/毫升）/0.1= 40毫克/毫升的聚（I:C）的最终浓度溶液。 警告:聚（I:C）粉末很轻。要小心，不要失去任何粉末，而从锅铲转移到锥形管。 </li><li>加0.9％氯化钠（盐水）相应体积成圆锥形桶e和盖上。直到解决方案是明确的:（C I）粉末通过在聚轻轻滚动滴生理盐水溶解的粉末。离心锥形管以800×g离心1分钟（ 图3C）。过滤聚（I:C）通过0.22微米的过滤器的解决方案。转移聚（I:C）溶液到1.5或2 ml离心管。可选:使用分光光度计来测量260/280比率的聚（I:C）的溶液中。确保该比率之间如制造商所描述1.54-1.82（ 图3）。注:（:C I）粉和作为对照注射应该是无菌的，药品级用于溶解聚生理盐水。 </li></ol> 6.盐水或聚（I:C）的E12.5注射<ol><li>称重秤上的鼠标并把它放回笼子。使用下面的公式来计算注射的体积为盐水和聚（I:C）小鼠:体重量（g）乘以5 =所需注射量（微升）。使用0.3毫升INSUL在注射器制订盐水的计算的体积或20毫克/公斤的聚（I:C）的解决方案， 例如，150微升在30 G鼠标。 </li><li>由尾根部轻轻拿起小鼠，并将其放置在笼顶部。轻拿轻放鼠标，以避免应力引起的混杂影响。插入针头，斜面向上，在大约20°相对于小鼠成其两个上乳头（ 图4）的中心，并注入盐水或聚（I:C）的溶液。注:针应该更换为注射后每只小鼠。 </li><li>将鼠标返回到其家笼。将笼子在一个安静，低流量的房间，以避免其他混杂的效果。 </li></ol> 7.聚后的第二天（I:C）注射（E13.5） <ol><li>检查以下早晨注射的小鼠。用规模来衡量体重。 注:聚（I:C）注射的孕鼠通常体重增加少于注射生理盐水注射上后的第二天小鼠挠度。 </li><li>直到后代是天生的不打扰老鼠。 </li></ol> 8.计MIA后代<ol><li>为了尽量减少MIA感应混杂影响，按照下面列出来衡量MIA后代的使用指南。 <ol><li>排除从窝早产或延迟生育，或者如果产仔数小于5。 </li><li>安乐死的CO 2的过度幼仔如果产仔大于10。 </li></ol></li></ol> 9.可选:检查急性炎症反应后MIA <ol><li>使用该机构的动物护理委员会批准的协议描述的方法注射:盐水或聚（C I）后牺牲孕鼠3小时。 注:颈椎脱臼往往是首选，因为它最大限度地减少CO 2 /安乐死药物对大坝和胚胎的影响。 </li><li>收集来自成年孕鼠脾脏和隔离胎盘和胎儿的胸罩在立体显微镜下。 <ol><li>对于脾虚收集，躺在解剖板鼠标和对孕鼠的腹部喷70％的乙醇。使用一对无菌剪刀，以使在腹部区域的胸腔下面通过皮肤中心的垂直切割。 </li><li>脾坐在左上腹部象限。使用镊子脾隔离开来。滚上微妙的任务雨刷脾，除去周围器官的脂肪组织。收集大约50毫克的脾组织并单独放置在Eppendorf管500微升RNA稳定缓冲区或TRIzol试剂，以防止核酸降解。储存在-80℃下冷冻样品直至使用。 </li><li>对于胎盘的收集，轻轻拔出从身体子宫角。放置子宫角在培养皿中填充有高压灭菌磷酸盐缓冲盐水（PBS），并离开在冰上的培养皿中。用两个镊子撕开子宫壁和揭露羊膜囊。 <ol><li>小心使用镊子打开羊膜囊不破坏胎盘和胎儿。分开胎儿胎盘和剪断脐带尽可能靠近胎盘越好。从胎盘中取出羊膜囊碎片。将胎盘分别在Eppendorf管500微升RNA稳定缓冲区或TRIzol试剂。储存在-80℃下冷冻直至使用的样本。 </li></ol></li><li>对于胎儿大脑的收集，储存在RNA从步骤9.2.2稳定缓冲区，直到解剖胎儿。从使用立体显微镜下微妙＃5镊子脑的脑室逗关闭软脑膜膜。小心地从胎儿大脑中删除所有的膜。将胎儿大脑单独在Eppendorf管500微升RNA稳定缓冲区或TRIzol试剂。储存在-80℃下冷冻直至使用的样本。 </li></ol></li><li>提取从组织的RNA和该RNA转变为cDNA。通过实际-T分析了基因的IL6基因表达IME PCR。 </li><li>由以下的TRIzol的制造协议提取组织中的RNA。如果组织被存储在RNA稳定化缓冲液中。解冻样品和降速的缓冲区。通过尖端转移组织到另一个微量离心用500μl的TRIzol并继续RNA提取。 </li><li>使用分光光度计来测量浓度，260/280及260/230比率RNA的。确保比率是2.0以上。 </li><li>消除由处理用DNase一混合物1微克的RNA的RNA，1微升10X反应缓冲液，1微升RNA酶的DNase，和无核酸酶水的DNA污染到的10微升的最终体积。孵育在37℃的主混合物30分钟。加入1μl的DNA酶终止溶液以终止反应。孵育在65℃下该混合物10分钟。 </li><li>反向转录RNA为cDNA。使用20微升的反应，以及高达1微克每反应的总RNA。混合4微升5倍的逆转录（RT）反应混合物缓冲液，1微升转ERSE酶，用DNase I和4微升无核酸-H 2 O处理后11微升RNA模板做出最后的20微升的解决方案。计划RT热循环的条件。一个通用的条件包括:第1步:25℃5分钟;步骤2:42℃30分钟;步骤3:85℃，5分钟;步骤4:保持在4℃。稀释的cDNA的5倍。对于短期储存，存放在4℃的基因。对于长期储存，冷冻在-20℃中的cDNA。 </li><li>分析中的cDNA通过实时PCR的IL6的基因表达和标准化IL6的基因表达对β-肌动蛋白基因的表达。 <ol><li>作出IL6和β-actin的检测预混液。使用25μl反应体系中，预混每个基因包含12.5微升的SYBR Green驴友，0.5微升10μM正向引物，0.5微升10μM反向引物和6.5微升无核酸酶的H 2 O的</li><li>将5微升5倍稀释的cDNA一Ť光学96孔反应板的孔的底部。吸取20μl反应混合液，每孔作最后的25微升PCR混合物。一式三份各基因对于每个样品。使用光学胶膜封板。在800 xg离心在4℃下使用标准的程序的实时PCR的降速板3分钟:步骤1:50℃，2分钟;步骤2:95℃，10分钟;步骤3:15秒和温度60℃下1分钟的95℃的40个循环。添加解离曲线的附加步骤。 </li></ol></li></ol> 10.检查在MIA成年后代的行为异常（可选）: <ol><li>在8-12周的年龄进行行为测试。改变笼的床上用品在测试前至少3天。适应小鼠的行为测试室测试前至少30分钟。 </li><li>对前脉冲抑制（PPI），抑制在有机玻璃圆筒小鼠与它下方的压电传感器。适应小鼠的PPI腔室5分钟。暴露小鼠120分贝白噪声（惊吓）的6项研究。 </li><li>然后用15分贝（1​​5 dB的背景噪声的14项试验，惊吓的14项试验，预脉冲5分贝（比背景噪声高5 dB为单位）+惊吓（PPI5）的14项试验和预脉冲的14项试验的随机混合物暴露小鼠比背景噪声+惊吓（PPI15）。 </li><li>平均为120dB的试验的第6试验的惊恐反应。酒店的平均其他个别刺激的惊吓反应。隐蔽的值前脉冲抑制（惊吓 - PPI5或PPI15）/惊吓。 </li><li>对于开放式现场测试中，将鼠标在一个开放的广场舞台（50×50×50 立方厘米 ）的角落。通过一个安装在天花板上的相机拍摄的小鼠10分钟的轨迹。定义竞技场为中心区域（17×17 平方厘米）的中心。量化行进的距离，中心区的条目，并且时间在中心区手动或通过基于图像的自动分析软件花费。 </LI&gt; <li>大理石埋藏测试，适应鼠标的测试笼压缩5厘米深，干净，阿斯松被褥10分钟。返回鼠标的居住笼。轻轻地放在20海军蓝直径1.5cm玻璃球中的4×5布置时尚。将鼠标回测试笼弹珠10分钟。算上已经被埋在10分钟的测试时间弹珠。 </li></ol> 11.检查小脑的神经病理学成人MIA后代（可选）: <ol><li>通过安乐死麻醉的盐水和MIA成年子女。灌注通过心血管系统用50ml PBS鼠标和随后50毫升4％多聚甲醛。 </li><li>小心取出大脑，并于4过夜postfix的在4％低聚甲醛的脑 C。冲洗大脑用PBS三次，并在PBS中的脑用0.02％叠氮化钠储存在4℃冰箱中直至使用。 注:后缀大脑可以存储在PBS中的0.02％草皮在冰箱IUM叠氮长达一个月。 </li><li>科脑入50微米的薄片矢状使用vibratome。收集用软毛笔的脑切片。通过温育在0.6％的过氧化氢的部分，在室温下30分钟终止该内源性过氧化物酶的活性。 </li><li>孵育在封闭缓冲液（在PBS中0.1％的Triton X-100和0.02％叠氮化钠的10％山羊血清）中稀释的抗钙结合蛋白抗体的部分在室温下过夜。然后孵育在对应于在室温下2小时封闭缓冲液稀释的生物素化的第二抗体的部分。 </li><li>孵育切片在抗生物素蛋白DH - 生物素化过氧化物酶H络合物缓冲器来检测生物素在室温下1小时。通过使用过氧化物酶底物以可视化的抗原开发的部分。中的步骤之间，用PBS洗涤切片三次。安装部分到显微镜载玻片。图像在显微镜下的章节。 </li></OL&gt;

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+immune+activation+alters+fetal+brain+development+through+interleukin-6.">Maternal immune activation alters fetal brain development through interleukin-6.</a> J Neurosci. 27 (40), 10695-10702 (2007).</li><li>Shi, L., 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=Activation+of+the+maternal+immune+system+alters+cerebellar+development+in+the+offspring.">Activation of the maternal immune system alters cerebellar development in the offspring.</a> Brain Behav Immun. 23 (1), 116-123 (2009).</li><li>Hsiao, E. Y., Patterson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+maternal+immune+system+induces+endocrine+changes+in+the+placenta+via+IL-6.">Activation of the maternal immune system induces endocrine changes in the placenta via IL-6.</a> Brain Behav Immun. 25 (4), 604-615 (2011).</li><li>Wu, W. L., 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+interaction+between+maternal+immune+activation+and+alpha+7+nicotinic+acetylcholine+receptor+in+regulating+behaviors+in+the+offspring.">The interaction between maternal immune activation and alpha 7 nicotinic acetylcholine receptor in regulating behaviors in the offspring.</a> Brain Behav Immun. , (2015).</li><li>Garay, P. A., Hsiao, E. Y., Patterson, P. H., McAllister, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+immune+activation+causes+age-+and+region-specific+changes+in+brain+cytokines+in+offspring+throughout+development.">Maternal immune activation causes age- and region-specific changes in brain cytokines in offspring throughout development.</a> Brain Behav Immun. 31, 54-68 (2013).</li><li>Garbett, K. A., Hsiao, E. Y., Kalman, S., Patterson, P. H., Mirnics, K. <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+maternal+immune+activation+on+gene+expression+patterns+in+the+fetal+brain.">Effects of maternal immune activation on gene expression patterns in the fetal brain.</a> Transl Psychiatry. 2, e98 (2012).</li><li>Hsiao, E. 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=Microbiota+modulate+behavioral+and+physiological+abnormalities+associated+with+neurodevelopmental+disorders.">Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders.</a> Cell. 155 (7), 1451-1463 (2013).</li><li>Naviaux, R. K., 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=Antipurinergic+therapy+corrects+the+autism-like+features+in+the+poly(IC)+mouse+model.">Antipurinergic therapy corrects the autism-like features in the poly(IC) mouse model.</a> PLoS One. 8 (3), e57380 (2013).</li><li>Pratt, L., Ni, L., Ponzio, N. M., Jonakait, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+inflammation+promotes+fetal+microglial+activation+and+increased+cholinergic+expression+in+the+fetal+basal+forebrain:+role+of+interleukin-6.">Maternal inflammation promotes fetal microglial activation and increased cholinergic expression in the fetal basal forebrain: role of interleukin-6.</a> Pediatr Res. 74 (4), 393-401 (2013).</li><li>Schwartzer, J. 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=Maternal+immune+activation+and+strain+specific+interactions+in+the+development+of+autism-like+behaviors+in+mice.">Maternal immune activation and strain specific interactions in the development of autism-like behaviors in mice.</a> Transl Psychiatry. 3, e240 (2013).</li><li>Khan, D., 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=Long-term+effects+of+maternal+immune+activation+on+depression-like+behavior+in+the+mouse.">Long-term effects of maternal immune activation on depression-like behavior in the mouse.</a> Transl Psychiatry. 4, e363 (2014).</li><li>Workman, A. D., Charvet, C. J., Clancy, B., Darlington, R. B., Finlay, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+transformations+of+neurodevelopmental+sequences+across+mammalian+species.">Modeling transformations of neurodevelopmental sequences across mammalian species.</a> J Neurosci. 33 (17), 7368-7383 (2013).</li><li>Abazyan, B., 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=Prenatal+interaction+of+mutant+DISC1+and+immune+activation+produces+adult+psychopathology.">Prenatal interaction of mutant DISC1 and immune activation produces adult psychopathology.</a> Biol Psychiatry. 68 (12), 1172-1181 (2010).</li><li>Meyer, U., 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=Adult+behavioral+and+pharmacological+dysfunctions+following+disruption+of+the+fetal+brain+balance+between+pro-inflammatory+and+IL-10-mediated+anti-inflammatory+signaling.">Adult behavioral and pharmacological dysfunctions following disruption of the fetal brain balance between pro-inflammatory and IL-10-mediated anti-inflammatory signaling.</a> Mol Psychiatry. 13 (2), 208-221 (2008).</li><li>Vuillermot, 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=Prenatal+immune+activation+interacts+with+genetic+Nurr1+deficiency+in+the+development+of+attentional+impairments.">Prenatal immune activation interacts with genetic Nurr1 deficiency in the development of attentional impairments.</a> J Neurosci. 32 (2), 436-451 (2012).</li><li>Bechard, A., Nicholson, A., Mason, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Litter+size+predicts+adult+stereotypic+behavior+in+female+laboratory+mice.">Litter size predicts adult stereotypic behavior in female laboratory mice.</a> J Am Assoc Lab Anim Sci. 51 (4), 407-411 (2012).</li><li>Harvey, L., 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=A+stereological+comparison+of+GAD67+and+reelin+expression+in+the+hippocampal+stratum+oriens+of+offspring+from+two+mouse+models+of+maternal+inflammation+during+pregnancy.">A stereological comparison of GAD67 and reelin expression in the hippocampal stratum oriens of offspring from two mouse models of maternal inflammation during pregnancy.</a> Neuropharmacology. 62 (4), 1767-1776 (2012).</li></ol>

The authors have nothing to disclose.

MIA感应在不同的时间窗扰乱在啮齿类动物不同脑发育的事件，并因此导致在后代不同行为异常和neuropathologies。这里，我们描述了一种协议以诱导MIA小鼠与聚（I:C）在E12.5注射。 MIA感应的此方法导致行为，神经病学，免疫学，并与自闭症和精神分裂症的后代8,10,11,16有关的胃肠道异常。要注意，这一点很重要，因为MIA是一个环境风险因素，它们的后代的表型，预计将有比来自神经发育障碍?...

母体免疫激活（MIA）的概念从母体感染患有自闭症和精神分裂症1的关联流行病学研究起源。由于没有在胎盘或母体病毒感染2,3后胎儿大脑检测复制病毒病原体，感染的对后代的影响是假设由母体免疫系统的激活，而不是病原体本身引起的。 为了阐明MIA和精神障碍，注入化学合成，病毒模拟双链RNA聚肌苷之间的原因和结果关系:胞苷酸:成怀孕啮齿类动物（聚（I C））已被广泛地用作动物模型MIA 4,5。聚（I:C）通过toll样受体3（TLR3），和聚的全身给药确认（I:C）诱导病毒样急性炎症反应。一种机制，通过它的聚（I:C）公关oduces行为异常和neuropathologies的后代是由母体胎盘胎儿轴6使亲和抗炎细胞因子的不平衡。一些研究小组已经通过在MIA模型理解精神障碍7的病因，并且由于研究组之间的不同的利益，免疫激活的不同时间点已被用来实现对脑发育和行为7不同扰动。 在保罗·帕特森实验室技术加州理工学院采用注射聚的策略（I:C）为孕鼠胚胎在12.5天（E12.5），它已经成功地证明了MIA能够诱导行为，神经的，并且在与自闭症和精神分裂症相关8-11后代的免疫学变化。我们之前的工作表明，MIA后代显示行为异常（ 如社会impairmenT，通信逆差，重复的行为，焦虑样行为，以及潜在抑制赤字8,10,12），免疫失调和细胞因子失衡8,13,14，胎儿大脑的基因表达15的改变，在小叶第七浦肯野细胞的损失小脑11，海马突触9性质改变，基因与环境互作13，肠道通透性的改变，以及肠道菌群组成16。此外，治疗和预防策略也从该模型系统13,16,17的发展。通过在E12.5诱导MIA，其他人已经表明MIA产生脑突触分子17的胎儿胶质细胞活化和前脑基底胆碱能18发育改造，株特异性相互作用19，大脑突触脑超微结构异常，脑线粒体呼吸链功能亢进abnormalties，下调，抑郁样行为，认知和海马长时程增强（LTP）障碍，与成人海马神经20的赤字。 在这里，我们提供了如何通过聚诱导MIA在E12.5的详细方法（I:C），以及如何运用这一模型来研究自闭症和精神分裂症的病因学范式。要注意的是MIA为多种病症4的危险因素是很重要的，其结果是对时间和感应的方法，以及在怀孕水坝的饲养极为敏感。这样，实验室之间甚至轻微不一致常常导致低的再现性和/或后代不同的表型。我们的方法是专门为那些有兴趣在学习MIA为自闭症和精神分裂症的环境风险因素设计的，详细的描述中提供将有助于研究人员提高他们的数据的可重复性。

<table><tbody><tr><td>Polyinosinic&amp;ndash;polycytidylic acid potassium salt</td><td>SIGMA</td><td>P9582</td><td></td></tr><tr><td>0.9% sodium chloride INJ. USP</td><td>HOSPIRA</td><td>NDC 0409-4888-10</td><td></td></tr><tr><td>MONOJECT insuline syrinage 3/10 ml 29 G x 1/2&quot;</td><td>COVIDIEN</td><td>8881600145</td><td></td></tr><tr><td>50 ml conical screw cap tubes</td><td>USA SCIENTIFIC</td><td>1500-1211</td><td></td></tr><tr><td>Nanodrop 1000 spectrophotometer</td><td>THERMO SCIENTIFIC</td><td>1000</td><td>Optional</td></tr><tr><td>Stereomicroscope</td><td>Wild Heerbrugg</td><td>M5A</td><td>Optional</td></tr><tr><td>Dumont #5 Forceps Inox Tip Size .10 X .06 mm</td><td>Roboz</td><td>RS-5045</td><td>Optional</td></tr><tr><td>RNAlater RNA stabilization reagent</td><td>Qiagen</td><td>76104</td><td>Optional</td></tr><tr><td>TRIzol reagent</td><td>Life Technologies</td><td>15596-026&amp;nbsp;</td><td>Optional</td></tr><tr><td>RQ1 Rnase-free DNase</td><td>Promega</td><td>M610A</td><td>Optional</td></tr><tr><td>iScript cDNA synthesis kit</td><td>Bio-Rad</td><td>170-8891</td><td>Optional</td></tr><tr><td>FastStart universal SYBR green master mix with ROX&amp;nbsp;</td><td>Roche</td><td>4913922001</td><td>Optional</td></tr><tr><td>Real-time PCR</td><td>ABI</td><td>7300</td><td>Optional</td></tr><tr><td>Primer: Il6 forward</td><td>Life Technologies</td><td>TAGTCCTTCCTACCCCAATTTCC</td><td>Optional</td></tr><tr><td>Primer: Il6 Reverse</td><td>Life Technologies</td><td>TTGGTCCTTAGCCACTCCTTC</td><td>Optional</td></tr><tr><td>Primer: beta-actin forward</td><td>Life Technologies</td><td>AGAGGGAAATCGTGCGTGAC</td><td>Optional</td></tr><tr><td>Primer: beta-actin Reverse</td><td>Life Technologies</td><td>CAATAGTGATGACCTGGCCGT</td><td>Optional</td></tr><tr><td>MicroAmp optical 96-well reaction plate</td><td>Life Technologies</td><td>4306737</td><td>Optionl</td></tr><tr><td>MicroAmp optical adhesive film&amp;nbsp;</td><td>Life Technologies</td><td>4311971</td><td>Optionl</td></tr><tr><td>EthoVision</td><td>Noldus</td><td>EthoVision</td><td>Optionl</td></tr><tr><td>SR-LAB apparatus (PPI)</td><td>San Diego Instruments&amp;nbsp;</td><td>SR-LAB</td><td>Optionl</td></tr><tr><td>Marbles</td><td>PENN-PLAX</td><td>Blue gem stones marbles</td><td>Optionl</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline (DPBS)</td><td>Life Technologies</td><td>21600-069</td><td>Optionl</td></tr><tr><td>Paraformaldehyde</td><td>MACRON</td><td>2621-59</td><td>Optionl</td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000 S</td><td>Optionl</td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td>Optionl</td></tr><tr><td>Triton x-100</td><td>Sigma</td><td>X100</td><td>Optionl</td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma</td><td>18312</td><td>Optionl</td></tr><tr><td>Goat serum</td><td>Vector Laboratories</td><td>S-1000</td><td>Optionl</td></tr><tr><td>Rabbit anti-calbindin antibody</td><td>Abcam</td><td>ab11426</td><td>Optionl</td></tr><tr><td>Biotinlyated goat anti-rabbit IgGantibody</td><td>Vector Laboratories</td><td>BA-1000</td><td>Optionl</td></tr><tr><td>VECTASTAIN ABC Kit</td><td>Vector Laboratories</td><td>PK-4000</td><td>Optionl</td></tr></tbody></table>

induction of maternal immune activation in mice at mid-gestation stage with viral mimic poly(i:c)

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric disorders. Numerous studies have demonstrated that MIA model is able to show face, construct, and predictive validity that are relevant to autism and schizophrenia. To model MIA, investigators often use viral mimic polyinosinic:polycytidylic acid (poly(I:C)) to activate the immune system in pregnant rodents. Generally, the offspring from immune activated dam exhibit behavioral abnormalities and physiological alterations that are associated with autism and schizophrenia. However, poly(I:C) injection with different dosages and at different time points could lead to different outcomes by perturbing brain development at different stages. Here we provide a detailed method of inducing MIA by intraperitoneal (i.p.) injection of 20 mg/kg poly(I:C) at mid-gestational embryonic 12.5 days (E12.5). This method has been shown to induce acute inflammatory response in the maternal-placental-fetal axis, which ultimately results in the brain perturbations and behavioral phenotypes that are associated with autism and schizophrenia.

<html>的20毫克/公斤的聚注射:在E12.5（I C）可以唤起在母体胎盘胎儿轴急性炎症反应并沉淀到大脑发育和行为表型12,13慢性效果。一个促炎性细胞因子水平升高，白细胞介素（IL）-6，是MIA后急性炎症反应的可靠指标。高峰时间在胎盘IL6的基因表达和胎儿大脑是在3小时后的聚（I:C）注射12,13。我们已经表明，聚（I:C）诱导整个脾脏产妇胎盘，胎儿的大脑...

Watch this Scientific Journal Video about Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C at JoVE.com

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C

母体免疫激活诱导小鼠的病毒性模仿聚妊娠中期阶段（I:C）

Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model.

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric ...

induction-maternal-immune-activation-mice-at-mid-gestation-stage-with

Research

JoVE Journal

Immunology and Infection

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic Poly(I:C)

18.2K 次观看.  California Institute of Technology. Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model.

视频: Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

产妇血管重构的评估在怀孕期间在小鼠子宫

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

发育生物学

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

使用<em&gt;在体内</em&gt;影像监测进展的实验小鼠巨细胞病毒感染的新生儿

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

免疫与感染

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

细胞因子产生的病毒逃避解剖先天免疫信号

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Mouse In Vivo Placental Targeted CRISPR Manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Mouse In Vivo Placental Targeted CRISPR Manipulation

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

小鼠 体内 胎盘靶向CRISPR操作

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients ...

遗传学

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

从小鼠组织在母胎界面分离白细胞

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

神经科学

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus (GBS)

Group B Streptococcus (GBS) is one of the most common bacteria isolated during human pregnancy. It is a leading cause of placental infection/inflammation, termed chorioamnionitis. Chorioamnionitis exposes the developing fetus to a high risk of organ injuries, perinatal morbidity, and mortality, as well as life-long neurobehavioral impairments and other non-neurological developmental issues. The two most frequent subtypes of GBS isolates from maternal and fetal tissues are serotypes Ia (13%-23%) and III (25%-53%). Our lab has developed and characterized a rat model of GBS-induced chorioamnionitis to study subsequent impacts on the central nervous system of the developing fetus and to understand underlying mechanistic aspects. This article presents the design as well as uses of the preclinical rat model, which closely reproduces the hallmark of GBS-induced chorioamnionitis in humans. This article aims to help scientists reproduce the experimental design as well as to provide support through examples of troubleshooting. The present model may also contribute to potential discoveries through uncovering causes, mechanisms, and novel therapeutic avenues, which remain unsettled in many developmental impairments arising from chorioamnionitis. Furthermore, the use of this model may be extended to the studies of perinatal non-neurological common and severe morbidities affecting, for instance, the retina, bowel, lung, and kidney. The main interest of this research is in the field of GBS-induced fetal neurodevelopmental impairments such as cerebral palsy (CP), attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD). The rationale supporting this model is presented in this article, followed by procedures and results.

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus GBS

The goal of this protocol is to describe a preclinical animal model of Group B Streptococcus (GBS)-induced chorioamnionitis. The study is designed to investigate mechanistic processes, potential causal links with developmental impairments, and finally to develop translational anti-inflammatory placento- and neuro-protective treatments.

Group B Streptococcus (GBS) is one of the most common bacteria isolated during human pregnancy. It is a leading cause of placental infection/inflammation, ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

胎儿接触产妇炎症的穆林模型，研究急性胆管炎对新生儿肠道发育的影响

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using Poly(I:C) to Study Susceptibility and Resilience in Offspring

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in offspring. Animal models of MIA are used to test causality, investigate mechanisms, and develop diagnostics and treatments for these disorders. Despite their widespread use, many MIA models suffer from a lack of reproducibility and almost all ignore two important aspects of this risk factor: (i) many offspring are resilient to MIA, and (ii) susceptible offspring can exhibit distinct combinations of phenotypes. To increase reproducibility and model both susceptibility and resilience to MIA, the baseline immunoreactivity (BIR) of female mice before pregnancy is used to predict which pregnancies will result in either resilient offspring or offspring with defined behavioral and molecular abnormalities after exposure to MIA. Here, a detailed method of inducing MIA via intraperitoneal (i.p.) injection of the double stranded RNA (dsRNA) viral mimic poly(I:C) at 12.5 days of gestation is provided. This method induces an acute inflammatory response in the dam, which results in perturbations in brain development in mice that map onto similarly impacted domains in human psychiatric and neurodevelopmental disorders (NDDs).

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using PolyI:C to Study Susceptibility and Resilience in Offspring

Maternal infection is a risk factor for neurodevelopmental disorders. Mouse models of maternal immune activation (MIA) may elucidate infection's impact on brain development and function. Here, general guidelines and a procedure are provided to produce reliably resilient and susceptible offspring exposed to MIA.

母体免疫激活诱导小鼠的病毒性模仿聚妊娠中期阶段（I:C）

摘要

探索更多视频

此视频中的章节

一个小鼠模型在子宫内移植

产妇血管重构的评估在怀孕期间在小鼠子宫

使用

细胞因子产生的病毒逃避解剖先天免疫信号

小鼠体内胎盘靶向CRISPR操作

从小鼠组织在母胎界面分离白细胞

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus (GBS)

胎儿接触产妇炎症的穆林模型，研究急性胆管炎对新生儿肠道发育的影响

使用聚（I:C）生成妊娠中期母体免疫激活的可重复模型，以研究后代的易感性和恢复力

母体免疫激活诱导小鼠的病毒性模仿聚妊娠中期阶段（I:C）

摘要

探索更多视频

此视频中的章节

一个小鼠模型在子宫内移植

产妇血管重构的评估在怀孕期间在小鼠子宫

使用

细胞因子产生的病毒逃避解剖先天免疫信号

小鼠 体内 胎盘靶向CRISPR操作

从小鼠组织在母胎界面分离白细胞

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus (GBS)

胎儿接触产妇炎症的穆林模型，研究急性胆管炎对新生儿肠道发育的影响

使用聚（I:C）生成妊娠中期母体免疫激活的可重复模型，以研究后代的易感性和恢复力

小鼠体内胎盘靶向CRISPR操作