由美国国立卫生研究院支持的授予R01-AA015614，R01-AA014973，T32-AA014127和K12-GM088021。作者感谢萨曼塔L.布基斯特技术援助和博士。凯文·考德威尔和唐纳德·帕特里奇的批判性评估的手稿和视频。

所有动物的程序批准了新墨西哥州，健康科学大学的中心机构动物护理和使用委员会。 1，蒸气室总成<ol><li>切聚碳酸酯板与用于顶部，底部，前，后，侧，和门设置在视频（ 图1和表1）的尺寸的圆锯或线锯。 </li><li>用圆锯或线锯，切于前面板的中间一个开口8英寸高16英寸宽。 </li><li>测量和由10英寸的聚碳酸酯片材，这将成为腔室的门上的孔的钢琴铰链马克到18英寸。 </li><li>在门口，钻埋头孔用5/16英寸的钻头，并用3/16英寸的钻头，钻孔的螺丝。确保钻埋头孔在门内侧的螺丝头。 </li><li>准备聚碳酸酯板进行焊接。 </li><li>来回组装NT，背面和侧面板使用上焊接上＃16的底部面板。 </li><li>密封板之间没有任何间隙与焊接式＃16。 </li><li>连接顶部面板，可焊＃16。 </li><li>使用杆夹持器或重课本持有一步到位，而焊缝治愈。 </li><li>通过一个12英寸的一块聚碳酸酯的附加一个1英寸到前面板1英寸的开口，用于门的铰链的安装如下。 </li><li>留出至少24小时的可焊治愈。 </li><li>切2片PEX管12英寸长，1片1英寸长的PEX管材的。 </li><li>用5/16的钻头，钻在每个12英寸PEX管材的约1至2英寸的孔从一端。 </li><li>附上2×12英寸的PEX管材的3/8英寸T型连接器与位于远离连接器的孔。 </li><li>盖上PEX管材的开口端用3/8英寸的插头。 </li><li>附加的钢琴铰链的前面板和门用4-40机器螺钉和螺母。 </li><li>使用门为指导，M方舟所需的钢琴铰链到所述腔室的前部面板上的1英寸的间隔件的孔中。 </li><li>用3/16英寸的钻头，钻孔插入前面板为钢琴铰链。 </li><li>安装门和钢琴铰链使用4-40机器螺钉和螺母的前面板。 </li><li>组装夹钳所示与上侧臂的任一侧上的垫圈和螺母的视频。 </li><li>马克和钻3/16英寸的孔为式夹钳在前面板上，并附加夹钳与4-40机器螺钉和螺母。 </li><li>加入3/8英寸橡胶球密封门的内侧。 </li><li>用5/8英寸无聊的钻头，钻在顶部面板的中心，进气口的孔。 </li><li>钻在背面板上的出口底部/中心有1/2英寸的孔。 </li><li>通过蒸汽室的所述出口孔的后壁推动的3/8英寸穿墙适配器的螺纹部分组装的出口端口。从安装螺母内室举行到位。 </li><li>从聚碳酸酯板取下保护盖。 </li><li>附加的1英寸片PEX管材的到T型连接器，并通过在从内侧腔室顶部的孔强制PEX管材。 </li><li>附加一个3/8英寸90弯头从外部的1英寸PEX管材的顶部。 注:钻在门上的1/2英寸的孔，并插入一个1/2英寸的密封隔成孔。 </li><li>重复步骤1-22仅供空气控制室。 </li></ol> 2，机架和空气输送大会<ol><li>如果放置室上在材料清单中提到的车/架，根据制造商的指示组装车。 </li><li>与备用螺母和螺栓，附加一个废料片聚碳酸酯到机架以保持气流调节器。 </li><li>任何地方的废料一块聚碳酸酯，标记和钻3/4英寸的孔为气流监管机构和监管部门重视与坚果提供。 </li><li>连接3/8英寸穿墙适配器与空气流量调节器的入口和出口。 </li><li>组装乙醇量瓶中，用曝气石，＃8挡和快速释放的在线连接。 </li><li>使用3/8英寸Tygon管，连接流量调节器，以使空气泵和乙醇的烧瓶， 如图1。 </li><li>用于空气只室中，附加的气流调节器的出口与入口3/8英寸90弯头用3/8英寸的聚乙烯管， 如图1。 </li></ol><img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51839/51839fig1highres.jpg" width="500" /> 的蒸气腔室的结构如图1所示。示意图。一个T型连接器连接到低噪声的空气泵。 T形连接器的一侧是直接连接到一个气流调节器的空气只有蒸汽室。在其他SIDE是再次分裂，并连接到两个不同的气流调节器，一个用于空气和1为乙醇。空气腔室直接连接到调节器，如图所示。对乙醇蒸汽室，一个气流调节器被连接到曝气石浸渍于液体乙醇中的过滤器的烧瓶中。过滤烧瓶的侧臂端口与空气调节器的输出连接所指示的。合并的乙醇蒸气与空气然后被连接到乙醇蒸气腔室的入口。出口管（未示出）被连接到在室内的换气口。 3，测试蒸汽房及调整色阶乙醇<ol><li>加600毫升190标准乙醇的过滤烧瓶中，将曝气石，挂钩到入口管向烧瓶的侧臂。 </li><li>关闭室门，并打开空气泵。 </li><li>调节气流调节器，使约一半的空气流过液体的醇，因为它是用混合的空气。 Adjus吨的空气只向空气流的乙醇室的酒精和空气的混合流。 </li><li>允许腔室，以测量空气乙醇浓度之前平衡至少30分钟。 </li><li>通过提取5毫升空气用18G针和60毫升注射器通过隔膜测量空气的乙醇浓度。通过绘制柱塞回〜60毫升（1:12稀释）稀释的样品与室内空气。按照制造商的指示使用呼吸测量空气乙醇水平。室内空气的稀释是需要在酒精的检测范围内实现酒精蒸汽的水平。 注意:如果使用不同级别的醇接触的稀释度可能需要进行调整。 </li><li>作为一个起点，因为需要达到约4.5-5克/分升（每克空气升汽化酒精）的空气酒精浓度调节气流调节器。 </li></ol> 4，动物育种<ol><li>女性群体内部C578BL / 6小鼠（2-3月龄）至少1周同步卵巢周期。 </li><li>单独房子雄性C57BL / 6小鼠（2-5月龄）至少2周。 </li><li>同步后，把一个单身女性与5天一个男丁，以便交配。 </li><li>以下交配，除去男性和女性单独房子并将其放置在室内。 </li></ol> 5，前和产后乙醇蒸气曝光<ol><li>暴露怀孕母畜乙醇蒸气每天4小时的光周期开始于上午10点（灯从早上6点 - 下午6点），除了诞生之日起，以防止小狗死亡。 </li><li>权衡对妊娠天（G）5，G13-G14，G18和-G20大坝监测怀孕;床上用品是天，女性进行称重，以减少搬运改变。 </li><li>每天更换食物的乙醇染毒组，以避免球团矿的消耗吸收到他们的任何乙醇。 </li><li>于年月日出生不公开的动物。出生后暴露母鼠和幼鼠，每天4小时开始时从P1-P12 10。 </li><li>权衡在P2，P8，P12，和P25幼仔;并更改P8和P12的床上用品，尽量减少额外的处理。 </li><li>紧随最后曝光（P12）后，将笼子转移到一个标准的动物房的房间。 </li></ol>

作者什么都没有透露。

在这里，我们将详细介绍方法的蒸气吸入室的建设。构建室所需要的材料和工具可容易地从多家商业供应商的步骤和腔室的结构相对简单。我们在这里描述的系统不包含任何直列式止回阀以防止反向流动和混合。我们无法测量任何酒精检测空气中的唯一腔说我们没有任何混合或乙醇回流到空气中只有室。只有腔室应定期检查对乙醇蒸汽的空气（应当指出的是，气室应始终乙醇室之前进行测试，以?...

怀孕期间饮酒可能会损害胎儿，造成持续性的改变在许多器官和系统，显著降低生活质量受影响的个人和家庭。据估计，女性约10〜30％的怀孕，在美国期间喝酒，在一个狂欢的模式1,2 1-8％饮用。胎儿发育过程中由乙醇暴露产生影响的范围内被统称为胎儿酒精谱系障碍（FASDs）。最近的估计表明，FASDs与患病率高达美国3 2-5％一个主要的公共卫生问题。 FASDs较严重的表现为胎儿酒精综合症（FAS），其特点是生长发育迟缓，颅面畸形和神经行为缺陷，包括学习障碍。 FAS的患病率估计为0.2-0.7％，在美国3。对于FASDs目前可用的治疗只是部分有效和更有效的治疗方法的发展是由这种复杂疾病的频谱的细胞和分子基础的认识不足的限制。 从全国出生缺陷预防研究（NBDPS）的数据表明，孕妇在第一孕期最常见的饮料，怀孕已检测前，中后妊娠2阶段其次是禁欲。 NBDPS接受还发现，乙醇消费量在妊娠期间的第二个最常见的模式涉及到整个怀孕2的所有孕期饮用。其原因包括缺乏认识胎儿酒精暴露（即使在低剂量），有限的访问产前检查，对神经精神疾病阳性家族史，以及滥用或酒精依赖4的潜在有害影响。有趣的是，NBDPS报道，消费的第三个最常见的模式在1禁欲参与<sup&gt;第一及第二学期其次是消费的第三季度，当它常常想当然地认为饮酒是安全的，因为器官已经大部分完成时。然而， 第三学期是一个时期高易感性乙醇引起的神经系统损害，因为这是当神经回路发生深刻的细化2期。 NBDPS接受还发现酒精消费在怀孕期间发生的其他不太常见的模式，包括整个第一和第二孕期其次是禁欲第三学期2时消耗。 在试图建模乙醇消耗量的不同图案的孕妇观察到，一些发育乙醇曝光范式已经建立了采用不同的动物物种，与大鼠和小鼠是最常见的5,6。怀孕在这些动物中的停留时间通常拉斯TS约3周，其对应于1 次和人类妊娠的第二个三个月。许多啮齿动物的研究已经评估不同剂量和乙醇曝光模式在这一时期的影响。通过液体的饮食7,8，加入乙醇到饮用水9,10，糖精增甜的解决方案的自愿饮用11，灌胃12，蒸气吸入13经常用于施用乙醇怀孕小鼠的方法和大鼠的例子包括管理和皮下或腹膜内注射14。这些研究的结果都概括几个在人类与FASDs观察到的赤字，在怀孕的早期阶段表明曝光足够整个大脑（在6,15审阅）损坏的神经元回路。 实验用啮齿类动物也显示出，等效于3 次时的曝光</sup&gt;人类妊娠，这大致对应于第一1-2周出生后的大鼠和小鼠中，中晚期可以显著影响大脑的发育。暴露在此期间进行了建模通过施用乙醇新生大鼠或小鼠。使用各种方法，包括通过胃造口术在人工饲养的动物16-18，胃内插管19， ​​皮下注射20馈送，并且蒸气吸入21,22乙醇已被施用给这些动物。这些研究令人信服地证明，大脑的井喷式增长是高发期易受乙醇6发育的影响。 正如上面提到的，在怀孕的各个孕期饮酒是酒精消费的女性2的通用模式。然而，相对很少有研究评估了利用动物模型曝光此模式的影响。一些研究已经采取advantagË大型动物，其中第三学期相当于在子宫内 ，而不是在新生儿期出现在大鼠和小鼠的情况下。这些动物模型包括非人类的灵长类23,24和羊25-27。然而，这些动物模型中没有得到广泛使用，因为成本高，需要专门的护理设施中FASDs研究，部分。鼠害已较常用的表征对胎儿发育5全孕期酒精暴露的影响。豚鼠在这方面给出了大脑的成熟其广泛的产前发展和相似之处，人类28,29的是特别有利的。用豚鼠，它有可能为乙醇曝光的效果刻画在子宫内 ，其中包括人类第三孕期的等效发展时期。这些动物中的相对较高的成本，以及其怀孕的时间比较长（〜67天），已经它们的使用仅限于工作FASDs研究的几个实验室。 因为在生物医学研究中的成本效益和广泛应用，研究者用大鼠在妊娠的各个孕期暴露于酒精模型。在最初的研究中，大鼠在怀孕期间通过流质饮食其次是乙醇行政胃经人工饲养的新生儿（出生后的天数（P）1-10），导致血峰乙醇含量（BEC）在0.08克/分升的大坝暴露而在幼崽0.16克/分升。这种模式引起持久的改变在视神经髓鞘形成和减少贝格曼神经胶质纤维在小脑30-32的数目。同样，使用在第三孕期当量（P4-9）33,34的一部分，通过胃内插管后新生儿的管理给予乙醇孕鼠水坝在狂欢般的方式人工饲养条件下迈尔和合作者。 PEAK孕产妇和小狗建筑物能源效益守则分别为0.3克/分升在两个孕20天，小六。这一切孕期曝光模式导致生长迟缓，这是显著大于在妊娠期间33选择的期间暴露幼仔观察。此外，大鼠相当于所有孕期期间暴露于乙醇中表现出小脑浦肯野和颗粒细胞，显着高于在动物其他时段34暴露观察到的数量的减少。在海马细胞数量的减少也报告了这一模式，但这种影响似乎主要是暴露的后果第三学期相当于35时。涉及行政乙醇通过灌胃既孕鼠及新生小鼠的方法也被用来模拟所有孕早期接触36。这种方法，取得了0.13克/分升的水坝（孕17天）和0.24克/分升小六的幼崽，引起离子的BEC克持久的改变中单胺神经递质的水平在海马和下丘脑，和DNA甲基转移酶和甲基CpG结合蛋白2在海马37,38的表达增加。使用类似的曝光模式（BEC = 0.14-0.2克/分升的水坝和0.2克/分升的幼崽），吉尔- Mohapel 等 39检测增加了新的未成熟的神经元在成年大鼠海马齿状回的数可能代表一种代偿机制，在成年出生的神经元的成熟乙醇诱导的神经元损伤或改变。研究者也试图透过液体饮食暴露水坝或怀孕和哺乳期间9,40饮水来模拟所有的孕期酒精暴露。然而，通过他们的母亲的乳汁露出幼崽的效用是有限的，因为它通常会导致较低的小狗建筑物能源效益守则（ 例如 ，0.002〜0.05克/分升; 41,42）。 小鼠也被用于EXTENS结构延续表征发育乙醇暴露的影响。这种动物模型股上述许多的大鼠动物模型，具有额外的优势的优势，许多转基因小鼠品系可用5。老鼠已经成功地使用了第一 ， 第二或怀孕43,44的第三学期中乙醇的效果来表征。然而，对这些动物的所有孕早期曝光的影响还没有得到很好的特点，因为它在技术上是比较难在相当于人类怀孕的各个孕期暴露小鼠。例如，人工饲养和灌胃，其已经成功地用于在大鼠中，需要在小鼠45更专门的程序。据我们所知，只有一个研究，迄今已尝试研究利用老鼠进行的所有孕期酒精暴露的影响;这些动物暴露于乙醇溶液中的饮用水杜里纳克怀孕和哺乳46。产妇的BEC分别为0.07克/分升和小狗的BEC没有确定，但预计将那些在堤坝的一小部分。 在这里，我们描述了其中的酒精是通过蒸气吸入室给予双方怀孕母畜和新生儿小鼠的所有孕期酒精暴露的新模式。根据先前的设计47蒸气室建成。我们提供了有关如何构建吸入室，开展曝光程序的详细说明。我们还提供对可达到的的BEC和暴露对幼鼠的存活和生长的影响的信息。

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construction of vapor chambers used to expose mice to alcohol during the equivalent of all three trimesters of human development

暴露于酒精在开发过程中可能导致的被统称为胎儿酒精谱系障碍（FASDs）的形态和行为异常的星座。在光谱的最严重的到底是胎儿酒精综合症（FAS），其特点是生长发育迟缓，颅面畸形学和神经行为缺陷。研究，动物模型，包括啮齿类动物，已经阐明参与FASDs的病理生理许多分子和细胞机制。乙醇处理对怀孕啮齿动物已被用来在怀孕的第一和第二孕期人体暴露模型。孕晚期乙醇消费量在人类已经使用新生儿啮齿类动物为蓝本。然而，很少有研究啮齿动物有特点乙醇暴露在相当于人类怀孕，曝光就是常见于孕妇的模式的所有三个学期的效果。在这里，我们将展示如何从随手打造蒸汽室Øbtainable材料，可以各容纳6个标准鼠笼。我们描述了一个蒸汽室的范例，可以用来模拟暴露于乙醇中，用最少的处理，在所有的3个三个月。我们的研究表明，怀孕母畜发展显著的代谢耐受乙醇。然而，新生小鼠没有发展代谢宽容和胎儿的数目，胎儿体重，胎盘重量，为幼崽/产仔数，死亡幼仔/产仔数和小狗体重并没有显著影响乙醇暴露。这种模式的一个重要优势就是它的适用性与转基因小鼠的研究。此外，这种模式最大限度地减少处理的动物，在胎儿酒精研究的一个主要变乱。

图2A显示了两个孕鼠及新生后代暴露于相对稳定的乙醇蒸气浓度在室内。这些范围。 图2B显示了在妊娠小鼠获得作为时间的函数的的BEC 4-6克/分升之间。的BEC使用标准的醇脱氢酶测定法基于48测量。在G5，能源效益守则迅速上升到〜60毫米2小时暴露后开始见顶，并在4小时暴露期结束。建筑物能源效益守则曝光结束后逐渐下降至〜12毫米的额外4小时后。由G13-14，有一...

Watch this Scientific Journal Video about Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development at JoVE.com

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

我们证明的使用，能同时容纳6鼠笼现成材料酒精蒸气室的建设。我们进一步描述了在胎儿酒精暴露的小鼠模型相当于人类怀孕的所有3个学期的使用。这一范式妊娠和产后1-12天期间暴露的动物。

蒸汽腔体建设用于公开小鼠酒精在所有三个学期人类发展的等效

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

construction-vapor-chambers-used-to-expose-mice-to-alcohol-during

Research

JoVE Journal

Medicine

14.6K 次观看.  University of New Mexico Health Sciences Center. 我们证明的使用，能同时容纳6鼠笼现成材料酒精蒸气室的建设。我们进一步描述了在胎儿酒精暴露的小鼠模型相当于人类怀孕的所有3个学期的使用。这一范式妊娠和产后1-12天期间暴露的动物。 

视频: Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

运用<em&gt;离体</em&gt;全胎儿各器官的直立滴文化的过程中鼠标研究器官发育过程

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

科研

发育生物学

Moderate Prenatal Alcohol Exposure and Quantification of Social Behavior in Adult Rats

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several independent laboratories have demonstrated robust alterations in the social behavior of rodents exposed to alcohol during brain development across a wide range of exposure durations, timing, doses, and ages at the time of behavioral quantification. Prior work from this laboratory has identified reliable alterations in specific forms of social interaction following moderate prenatal alcohol exposure (PAE) in the rat that persist well into adulthood, including increased wrestling and decreased investigation. These behavioral alterations have been useful in identifying neural circuits altered by moderate PAE1, and may hold importance for progressing toward a more complete understanding of the neural bases of PAE-related alterations in social behavior. This paper describes procedures for performing moderate PAE in which rat dams voluntarily consume ethanol or saccharin (control) throughout gestation, and measurement of social behaviors in adult offspring.

The goal of the protocol presented here is to describe procedures to expose rats to moderate levels of alcohol during prenatal brain development and to quantify resulting alterations in social behavior during adulthood.

适度的产前酒精暴露在成年大鼠社会行为的量化

Alterations in social behavior are among the major negative consequences observed in children with Fetal Alcohol Spectrum Disorders (FASDs). Several ...

行为学

Wheel Running and Environmental Complexity as a Therapeutic Intervention in an Animal Model of FASD

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, such as rates of adult neurogenesis, angiogenesis, and expression of neurotrophic factors in rodents. This intervention has also been shown to mitigate behavioral and neuroanatomical aspects of the negative impacts of teratogens (i.e., developmental exposure to alcohol) and age-related neurodegeneration in rodents. Environmental complexity (EC) has been shown to produce numerous neuroplastic benefits in cortical and subcortical structures and can be coupled with wheel running to increase the proliferation and survival of new cells in the adult hippocampus. The combination of these two interventions provides a robust &#34;superintervention&#34; (WR-EC) that can be implemented in a range of rodent models of neurological disorders. We will discuss the implementation of WR/EC and its constituent interventions for use as a more powerful therapeutic intervention in rats using the animal model of prenatal exposure to alcohol in humans. We will also discuss which elements of the procedures are absolutely necessary for the interventions and which ones may be altered depending on the experimenter&#39;s question or facilities.

Cardiovascular exercise and stimulating experiences in a complex environment have positive benefits on multiple measures of neuroplasticity within the rodent brain. This article will discuss the implementation of these interventions as a &#34;superintervention&#34; which combines wheel running and environmental complexity and will address the limitations of these interventions.

车轮运行环境的复杂性在胎儿酒精中毒综合症的动物模型干预治疗

Aerobic exercise (e.g., wheel running (WR) extensively used in animal research) positively impacts many measures of neuroplastic potential in the brain, ...

Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This is mostly due to the difficulty of microdissection of fetal brains and their sectioning for determination of apoptotic cells caused by prenatal exposure to alcohol. The experiments described here provide visualized techniques from mice breeding to the identification of cell death in fetal brain tissue. This study used C57BL/6 mice as the animal model for studying fetal alcohol exposure and the role of trophic peptide against alcohol-induced apoptosis. The breeding consists of a 2-hr matting window to determine the exact stage of embryonic age. An established fetal alcohol exposure model has been used in this study to determine the effects of prenatal alcohol exposure in fetal brains. This involves free access to alcohol or pair-fed liquid diets as the sole source of nutrients for the pregnant mice.
The techniques involving dissection of fetuses and microdissection of fetal brains are described carefully, since the latter can be challenging. Microdissection requires a stereomicroscope and ultra-fine forceps. Step-by-step procedures for dissecting the fetal brains are provided visually. The fetal brains are dissected from the base of the primordium olfactory bulb to the base of the metencephalon.
For investigating apoptosis, fetal brains are first embedded in gelatin using a peel-away mold to facilitate their sectioning with a vibratome apparatus. Fetal brains embedded and fixed in paraformaldehyde are easily sectioned, and the free floating sections can be mounted in superfrost plus slides for determination of apoptosis or cell death.
TUNEL (TdT-mediated dUTP Nick End Labeling; TdT: terminal deoxynucleotidyl transferase) assay has been used to identify cell death or apoptotic cells. It is noteworthy that apoptosis and cell-mediated cytotoxicity are characterized by DNA fragmentation. Thus, the visualized TUNEL-positive cells are indicative of cell death or apoptotic cells.
The experimental designs here provide information about the use of an established liquid diet for studying the effects of alcohol and the role of neurotrophic peptides during pregnancy in fetal brains. This involves breeding and feeding pregnant mice, microdissecting fetal brains, and determining apoptosis. Together, these visual and textual techniques might be a source for investigating prenatal exposure of harmful agents in fetal brains.

The experimental designs proposed here focus on studying the effects of alcohol exposure in apoptosis and the application of neurotrophic peptide during pregnancy in fetal brain. A detailed description from the breeding to the collection of fetal brains is described. Techniques for determination of apoptosis are also described in detail.

实验方法测试的影响，在怀孕期间对酒精诱导的细胞凋亡在C57BL / 6小鼠神经营养肽，ADNF-9

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This ...

神经科学

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

使用头太空气色谱对斑马鱼胚胎中的乙醇水平进行定量

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

酒精减少芥末反应:小鼠的行为和 AI 分析

Integration of Animal Behavioral Assessment and Convolutional Neural Network to Study Wasabi-Alcohol Taste-Smell Interaction

The commercial wasabi pastes commonly used for food preparation contain a homologous compound of chemosensory isothiocyanates (ITCs) that elicit an irritating sensation upon consumption. The impact of sniffing dietary alcoholic beverages on the sensation of wasabi spiciness has never been studied. While most sensory evaluation studies focus on individual food and beverages separately, there is a lack of research on the olfactory study of sniffing liquor while consuming wasabi. Here, a methodology is developed that combines the use of an animal behavioral study and a convolutional neural network to analyze the facial expressions of mice when they simultaneously sniff liquor and consume wasabi. The results demonstrate that the trained and validated deep learning model recognizes 29% of the images depicting co-treatment of wasabi and alcohol belonging to the class of the wasabi-negative liquor-positive group without the need for prior training materials filtering. Statistical analysis of mouse grimace scale scores obtained from the selected video frame images reveals a significant difference (P &lt; 0.01) between the presence and absence of liquor. This finding suggests that dietary alcoholic beverages might have a diminishing effect on the wasabi-elicited reactions in mice. This combinatory methodology holds potential for individual ITC compound screening and sensory analyses of spirit components in the future. However, further study is required to investigate the underlying mechanism of alcohol-induced suppression of wasabi pungency.

作者聚焦:探索味觉-嗅觉相互作用 - 使用酒精气味缓解芥末灼伤

This article describes a set of methods for measuring the suppressive ability of sniffing alcoholic beverages on the wasabi-elicited stinging sensation.

动物行为评估与卷积神经网络相结合研究芥末-酒精味-嗅相互作用

The commercial wasabi pastes commonly used for food preparation contain a homologous compound of chemosensory isothiocyanates (ITCs) that elicit an ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

使用的跟踪经典条件反射，评估胎儿酒精谱系障碍大鼠模型海马功能障碍

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

蒸汽腔体建设用于公开小鼠酒精在所有三个学期人类发展的等效

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适度的产前酒精暴露在成年大鼠社会行为的量化

车轮运行环境的复杂性在胎儿酒精中毒综合症的动物模型干预治疗

实验方法测试的影响，在怀孕期间对酒精诱导的细胞凋亡在C57BL / 6小鼠神经营养肽，ADNF-9

小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价

使用头太空气色谱对斑马鱼胚胎中的乙醇水平进行定量

动物行为评估与卷积神经网络相结合研究芥末-酒精味-嗅相互作用

动物行为评估与卷积神经网络相结合研究芥末-酒精味-嗅相互作用

动物行为评估与卷积神经网络相结合研究芥末-酒精味-嗅相互作用

使用的跟踪经典条件反射，评估胎儿酒精谱系障碍大鼠模型海马功能障碍