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=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 border="0" cellpadding="0" cellspacing="0" width="711">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Polyinosinic&#8211;polycytidylic acid potassium salt</td>
			<td style="width:129px;">SIGMA</td>
			<td style="width:167px;">P9582</td>
			<td style="width:133px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9% sodium chloride INJ. USP</td>
			<td>HOSPIRA</td>
			<td>NDC 0409-4888-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MONOJECT insuline syrinage 3/10 mL 29G x 1/2&#34;</td>
			<td>COVIDIEN</td>
			<td>8881600145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml conical screw cap tubes</td>
			<td>USA SCIENTIFIC</td>
			<td>1500-1211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanodrop 1000 spectrophotometer</td>
			<td>THERMO SCIENTIFIC</td>
			<td>1000</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereomicroscope</td>
			<td>Wild Heerbrugg</td>
			<td>M5A</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Forceps Inox Tip Size .10 X .06mm</td>
			<td>Roboz</td>
			<td>RS-5045</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAlater RNA stabilization reagent</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIzol reagent</td>
			<td>Life Technologies</td>
			<td>15596-026&#160;</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RQ1 Rnase-free DNase</td>
			<td>Promega</td>
			<td>M610A</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iScript cDNA synthesis kit</td>
			<td>Bio-Rad</td>
			<td>170-8891</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastStart universal SYBR green master mix with ROX&#160;</td>
			<td>Roche</td>
			<td>4913922001</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Real-time PCR</td>
			<td>ABI</td>
			<td>7300</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primer: Il6 forward</td>
			<td>Life Technologies</td>
			<td>TAGTCCTTCCTACCCCAATTTCC</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primer: Il6 Reverse</td>
			<td>Life Technologies</td>
			<td>TTGGTCCTTAGCCACTCCTTC</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primer: beta-actin forward</td>
			<td>Life Technologies</td>
			<td>AGAGGGAAATCGTGCGTGAC</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primer: beta-actin Reverse</td>
			<td>Life Technologies</td>
			<td>CAATAGTGATGACCTGGCCGT</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MicroAmp optical 96-well reaction plate</td>
			<td>Life Technologies</td>
			<td>4306737</td>
			<td>Optionl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MicroAmp optical adhesive film&#160;</td>
			<td>Life Technologies</td>
			<td>4311971</td>
			<td>Optionl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EthoVision</td>
			<td>Noldus</td>
			<td>EthoVision</td>
			<td>Optionl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SR-LAB apparatus (PPI)</td>
			<td>San Diego Instruments&#160;</td>
			<td>SR-LAB</td>
			<td>Optionl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Marbles</td>
			<td>PENN-PLAX</td>
			<td>Blue gem stones marbles</td>
			<td>Optionl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Phosphate-Buffered Saline (DPBS)</td>
			<td>Life Technologies</td>
			<td>21600-069</td>
			<td>Optionl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>MACRON</td>
			<td>2621-59</td>
			<td>Optionl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td>Leica</td>
			<td>VT1000 S</td>
			<td>Optionl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td>Optionl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton x-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>Optionl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrogen peroxide solution</td>
			<td>Sigma</td>
			<td>18312</td>
			<td>Optionl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat serum</td>
			<td>Vector Laboratories</td>
			<td>S-1000</td>
			<td>Optionl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit anti-calbindin antibody</td>
			<td>Abcam</td>
			<td>ab11426</td>
			<td>Optionl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinlyated goat anti-rabbit IgGantibody</td>
			<td>Vector Laboratories</td>
			<td>BA-1000</td>
			<td>Optionl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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)

18.1K 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

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

SJL小鼠诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

科研

免疫与感染

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

右心室收缩压测量组合在小鼠的肺和免疫组织样品的收获

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

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. ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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 ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

确定免疫系统抑制中枢神经系统对保护药物干预自身免疫性脱髓鞘

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...