Name: assessing cellular stress and inflammation in discrete oxytocin-secreting brain nuclei in the neonatal rat before and after first colostrum feeding
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作者感谢马农·游侠和亚历山大·舒尔茨在编写本议定书方面提供的协助。

这项研究得到了哥伦比亚大学动物护理和使用机构委员会和纽约州精神病研究所的批准。
1. 组织制备
<ol><li>从供应商处订购怀孕的老鼠。</li>
	<li>跟随定时怀孕的老鼠, 观察它们到达后的几周内生长的腹部, 然后在预期的分娩日期通过每2小时检查一次笼子, 直到分娩开始, 寻找幼崽。</li>
	<li>在未引物的幼崽 (在观察腹部时没有明显的白色乳腹) 之前, 或在为前提狗 (此时在它们...

<ol>
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本文介绍了一种在新生大鼠脑内对离散、富 or 脑核进行微解剖的技术。人们公认, 神经元是高度专业化的, 即使在大脑中的良好特征的细胞核内也是如此。这种高重现性的方法可以分离出特定的富含 otr 的原子核, 从而能够进行可靠的假设检验。利用西方的自动印迹, 进一步提高了结果的一致性和重现性。虽然这种技术的局限性仍然是适度的大脑冲床变异性;这项技术代表了比单细胞、整个大脑或大?...

与我们对产后白天至几周大脑早期发育的理解不同, 对老鼠生命最初几个小时发生的无数动态变化的了解相对较少。一个关键的挑战是新生大鼠大脑体积小, 以及需要高科技工具来分离离散的大脑区域或单个细胞。研究通常评估基因转录, 而不是翻译1,2, 这不能使一个坚实的理解功能水平的激活信号分子。另一些人使用免疫组织化学来检查表达, 以参考大脑区域, 这不允许量化表达水平3。到目前为止, 还没有研究过与大鼠在离散大脑区域的第一初乳饲料相关的信号通路的激活, 这需要用西方方法快速分离和牺牲和测量蛋白质表达和蛋白质磷酸化杂交。虽然大脑微解剖是在年龄较大的大脑上进行的, 但我们还没有确定在 p0 大脑中进行非单细胞脑拳的参考。本文提出了一种利用相对低技术的打孔技术和西方印迹技术分离新生儿大脑受限区域的方法, 以测量相对较小样本中的蛋白质表达。该协议可能适用于需要评估任何物种小大脑的相对受限区域的蛋白质表达和翻译后修饰 (例如磷酸化) 的研究问题, 前提是用户可以通过地图集和可识别的地标直观地识别感兴趣的大脑区域。
这项技术是为了了解新生大鼠的第一牛初乳饲料所发生的变化而开发的, 这种饲料富含催产素 (ot)。长期以来, ot 一直以其刺激牛奶脱床和子宫收缩的能力而闻名。然而, ot 现在被认为在调节许多身体功能和行为方面发挥着广泛的作用.例如, ot 反对压力和炎症, 再加上适应性从属行为5, 延迟胃排空, 减缓肠道转运。ot 受体 (otr) 已被鉴定在肠内神经元和肠道上皮 6, 7,8.ot 对婴儿产后早期的胃肠道效应尤为重要。例如, 母乳喂养与向新生儿肠道9、10 提供大量 ot 有关, 数据显示, 在牛奶乳汁期间, otr 在十二指肠绒毛中严重过度表达.
利用肠道细胞系进行的体外实验在细胞水平上证明, 催产素调节了压力信号通路11、12中的重要分子, 并在蛋白质的转化中发挥了调控作用12. 这些研究表明, 牛奶的成分, 包括来自母亲的外源性催产素, 对新生儿展开的蛋白质反应非常重要, 以减少细胞压力 13.
体内和体外研究表明, 牛初乳 ot 调节新生大鼠肠道绒毛的细胞应激反应、炎症和自体吞噬标志物。当肠道同时暴露在14天初乳14、15 和许多蛋白质中的母亲的微生物群中时, 新生儿肠细胞的腔侧承受着巨大的细胞压力, 其中包括 ot9等激素,10,16岁
ot 对大脑的影响已经研究了17。然而, 在产后早期肠道中显示的 ot 信号机制还没有在大脑中得到研究。本文采用电泳方法对新生大鼠脑干和下丘脑离散脑核进行了隔离。这种方法的总体目标是捕捉大脑区域的细胞信号状态, 尽可能接近出生, 在第一次牛奶乳汁之前和之后, 在胶质神经元指数最低的脑组织中。开发这项技术的理由是, 它可以快速分离新生幼崽中受限制的、微观的大脑区域, 并使用西方的自动印迹进行更均匀的神经元收集, 用于体外研究方法, 在相对较小的解剖样本上提供高度一致的结果。先前工作的一个缺点包括更多的严重解剖 (大脑切片或整个大脑) 和年龄较大的动物 18,19。幼崽的大脑是令人难以置信的动态, 具有出生后的胶质分化波。为了研究幼崽首次进食对大脑变化的影响, 有必要研究具有可重复解剖的受限神经元核。
牛奶饲料通常被分析它的免疫学和营养对健康或基因表达的影响 (例如, 在肠细胞20,21), 而它的影响对大脑区域在脑子发展过程中很少被研究。结合肠道胆囊激素受体对脑干核的迁移, 而不是细胞内信号通路 22, 分析了牛奶在肠道中的转运对大脑功能的影响。关于发育中的新生儿大脑在怀孕 23岁期间容易营养不良, 母亲营养不良的文献很多, 但压力和炎症信号没有得到解决。重要的是, 目前的方法利用了一种现象, 在一天零大鼠新生儿分离血液出生的牛初乳刺激从迷迷神经中继的内脏刺激。这就是所谓的应力低反应期, 其特征是出生后立即未成熟的孤束核 (nts)-下丘脑电路 24, 25 限制 nts, 室旁核 (pvn),超视核 (son) 信号到血液出生的刺激。
这种方法对于分析多种信号通路和相对限于神经元细胞是有用的, 前提是在大鼠产后第0天采集脑组织, 以及母亲是否受到任何形式的治疗在怀孕期间。可以分析垃圾对初乳饲料与预喂养信号的影响。当比较大脑区域与蛋白质产量低或丰富的蛋白质产量之间的信号时, 这种方法使毛细血管内确定毛细血管中多肽带的总蛋白与蛋白质抗原的免疫定量平行。该方法可以使用任意单位对没有标准定量曲线的相同抗体获得的结果进行定量比较, 并参照每毛细管的总蛋白。只有使用定量的标准曲线, 才有可能对不同抗体获得的结果进行比较。
这种方法可以评估肠道和大脑之间发生的双向信号, 这可以影响两个器官26的功能.催产素与食物摄入之间的联系,近年来已被广泛研究 27, 支持增加催产素信号和营养供应之间的联系。这些研究也支持相反的概念, 即能量不足与下丘脑催产素信号的减少相结合。
早期对 ot 对大脑活动的影响的研究表明, 诱导的肠道炎症引起下丘脑 pvn、杏仁核和梨形皮质的 cfos 转录, 可难治性于迷走神经切开术28。然而, 全身输注 ot 与分泌物减少脑 cfos 反应引起的炎症反应在肠道28。这表明外源 ot 的作用是通过迷速继电器以外的途径进行的, 可能是通过 6,29后携带的血液传播信号分子进行的。
在这项研究中, 对此前在肠道中观察到的细胞应激信号通路进行了大脑评估。假设是, 牛奶成分可以保护或推迟炎症对肠道对微生物和其他代谢物的渗透性的影响, 进而对大脑功能的影响。在13例牛初乳启动前后发现的 ikb 与 bi-p 信号的明显拮抗差异表明, 仍在发育过程中的新生儿大脑可能会感觉到这些牛初乳诱导的肠道信号。
测量了以往肠道实验中使用的与内质网应力相关的信号蛋白标记。它们包括 er 伴侣 bip、翻译起始因子 eif2a (用作应激反应积分器30)、eif2a 激酶 p-pkr 和两种炎症信号蛋白 (nf-kb 及其抑制剂 ikb)。
根据成人分泌或反应 ot 的能力, 选择了六个大脑区域。nts 位于上髓质, 是内脏输入的第一个中继, 通过相邻的区域后雷马32接收来自肠道内迷变神经感觉神经元的直接信号, 可能还有血液出生的细胞因子、毒素和激素.pvn、视上核 (son)、纹状体核 (str)、大脑皮层 (cx) 和内侧视前核 (mpo) 通过 nts 接收来自肠道的信号。
结果表明, nts 在初乳启动前和首次喂养后立即发生的产后细胞应激反应与 pvn 和 son 不同。cx、str 和 mpo 中的信令也不同于 pvn 和 son。先前显示的调节肠道细胞压力和炎症的 ot 的独特保护功能很可能是大脑某些区域所能感觉到的。总体而言, 数据表明, 在细胞水平上, 在出生后的最初几个小时内, 大脑会对与营养功能不足相关的代谢压力做出反应。数据还显示, 牛初乳饲料调节效果的范围和方向是区域依赖性的, 在一些地区, 它们反映了以前在肠道中显示的 ot 效应。

<table>
	<tbody>
		<tr>
			<td>Bradford solution</td>
			<td>Bio Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protein lysis kit</td>
			<td>Protein simple</td>
			<td>CBS403</td>
			<td>Bicine/CHAPS</td>
		</tr>
		<tr>
			<td>WES kits</td>
			<td>Protein simple</td>
			<td>WES-Mouse 12-230 master kit (PS-MK15), WES-Rabbit 12-230 master kit (PS-MK14), WES 12-230 kDa total Protein master kit (PS-TP07)</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse IgG HRP conjugate</td>
			<td>Protein simple</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-phospho-eIF2a</td>
			<td>Cell Signaling technology</td>
			<td>SER51, 9721</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse mAb anti-PKR</td>
			<td>Cell Signaling technology</td>
			<td>2103</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-phospho-PKR</td>
			<td>Millipore</td>
			<td>Thr451, 07-886</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit mAb anti-PKR</td>
			<td>Cell Signaling technology</td>
			<td>12297</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit mAb anti-GAPDH</td>
			<td>Cell Signaling technology</td>
			<td>2118</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse mAb anti-phospho-IKB</td>
			<td>Cell Signaling technology</td>
			<td>9246</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse mAb anti-IKB</td>
			<td>Cell Signaling technology</td>
			<td>4814</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit anti-BiP</td>
			<td>Cell Signaling technology</td>
			<td>3183</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti GCN2</td>
			<td>Cell Signaling technology</td>
			<td>3302</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit mAb anti-phospho-GCN2</td>
			<td>BIORBYT</td>
			<td>T899</td>
			<td></td>
		</tr>
		<tr>
			<td>pregnant Sprague-Dawley rats</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Punch device</td>
			<td>WellTech Rapid Core or Harris Uni-Core</td>
			<td>0.35, 0.50, 0.75, 1.0, 1.20, 1.50</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing cellular stress and inflammation in discrete oxytocin-secreting brain nuclei in the neonatal rat before and after first colostrum feeding

该方案的目标是在首次初乳喂养前后分离新生儿大脑中富含催产素受体的大脑细胞核。已知对代谢应激有反应的蛋白质的表达是在脑核分离株中使用西方印迹进行的。这样做是为了评估代谢应激引起的体内营养功能不全是否引发神经元应激。我们以前已经证明, 新生儿营养不足会引起肠道的代谢压力。此外, 初乳催产素可调节新生大鼠肠道绒毛首次进食前后的细胞应激反应、炎症和自体吞噬标志物。与内质网应力相关的信号蛋白标记 [er 伴侣结合免疫球蛋白蛋白 (bip)、真核翻译起始因子 2a (eif2a) 和 eif2a 激酶蛋白激酶 r (p-pkr)] 以及2炎症信号蛋白 [核因子-b (nf-kb) 和抑制剂 b (ikb)], 在新生脑核 [孤立道 (nts)、旁细胞核 (pvn)、超视核 (son)、皮层 (cx)、纹状体 (str) 和在第一次喂养 (初乳未启动) 之前和护理开始后 (由初乳启动) 之前的内侧视前细胞核 (mpo)。在未启动和下调的 nts 组织中, bip-grp78 和 pe-eif2a 的表达得到了抑制。nf-行在 cx、str 和 mpo 细胞质中保留 (高), 而 nf-kob 在 nts、pvn 和 son 中均较低且不变。集体 bip 和 p-eif2 的发现与应激反应是一致的。eif2a 在 son、cx、str 和 mpo 中由 dsrna 依赖性激酶 (p-pkr) 磷酸化。然而, 在 nts (在较小程度上在 pvn 中), eif2a 被另一种激酶磷酸化, 一般控制非接触-2 激酶 (gcn2)。以前在新生儿肠道细胞中观察到的压力调节机制似乎反映在一些富含 otr 的大脑区域。nts 和 pvn 可利用其他地区不同的磷酸化机制 (营养不足下), 并可耐火性的营养不足的影响。综合来看, 这些数据表明, 大脑对营养功能不全应激的反应被来自牛初启动的肠细胞的信号所抵消。

免疫反应带相对于总蛋白质表明, 有大脑细胞核的收获蛋白很低。这就需要使用西方的自动印迹技术, 与典型的西方印迹相比, 这种技术非常敏感。与西方印迹中的每车道相比, 这种方法可以用每毛细管少四倍的蛋白质运行。
初乳启动对脑核 bip 水平的差异效应
大鼠大脑的冠状部分是?...

Watch this Scientific Journal Video about Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding at JoVE.co

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

新生儿初乳喂养前后离散催产素分泌脑核细胞应激与炎症的评估

在这里, 我们提出了一个方案, 以分离脑核在新生大鼠大脑与第一初乳喂养。这项技术允许研究由肠细胞信号调节的大脑营养功能不足应激。

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

这种方法可以帮助回答产后发展领域关于牛奶吸乳如何影响大脑中压力信号的关键问题。这种技术可用于在第一个自然饥饿期捕获特定的大脑区域，并将它们与第一次自然初乳喂养后不久的各区域进行比较。了解吸吮如何影响行为和情绪的生理，在外活脑组织分析期间，能够对各种受体使用拮抗剂。为了获得核拳，用戴手套的手用尾巴把老鼠幼崽除掉。收获大脑后，在室温下迅速将整个器官放在聚甲基丙烯酸酯脑模中，并立即使用新的剃须刀刀片制作500微米厚的组织切片。将切片在培养皿中铺入烤盘，以保持各部分的正确方向。当整个大脑被切开后，迅速将不含葡萄糖的人工脑脊髓液加入到盘中，并在28至30摄氏度下孵育切片60分钟，并在轨道摇床上不断搅拌。使用大脑图集和每个组织上的解剖地标来识别要打孔的脑核，并在解剖显微镜下将感兴趣的核与感兴趣的核放在新的培养皿中。可视化后，使用皮质工具快速冲出四到六个不同的核，并快速将每个冲孔核浸入 0.06 mL 的冰冷蛋白提取缓冲液中，在适当标记的微离心管中，含有蛋白酶抑制剂和磷酸酶抑制剂，60 分钟。在孵化结束时，将提取的核在冷却的微型离心机中离心，并使用微移液器小心地将0.055 mL的上清液从每个管中转移到冰上适当的预冷却1.5 mL微型离心机管中。在负20摄氏度储存之前，将0.012 mL的上清液从每个蛋白质储存管转移到适当的0.5 mL冰上预冷却管中，进行第一次样品制备。要准备毛细管内蛋白质测量的样品，在 0.5 mL 标记管的每个样品中加入 0.003 mL 的蛋白质解菌试剂。接下来，在0.016 mL的去电化水中将0.004 mL的主混合试剂加入生物素化分子量梯溶液中，并在95摄氏度的热块中使梯子和蛋白质提取物样品变性，5分钟后将所有管在4摄氏度内储存。在信号蛋白分析方面，将0.01 mL新鲜准备好的过氧化物溶液加入新型自动西板E-1至E-25，向D-2至D-25井添加0.01 mL二级抗小鼠抗体，并将0.01 mL的streptavidin马萝卜易腐酶从试剂盒添加到井D-1。将0.01 mL新鲜准备好的原抗体加入到C-2至C-25的井中，将0.01 mL抗体稀释剂两种溶液加入到B-25井中。将 F 行井留空，用 0.45 mL 的洗涤缓冲液填充 F 行下方三行的五个隔间。接下来，将冷冻样品短暂旋转 30 秒，并将每个样品的 0.03 mL 添加到 A 行的每个井中，从 A-2 开始。然后，将0.005 mL的生物基化分子量梯添加到井 A-1中，并覆盖板进行离心。当板离心时，在自动化的西方相关软件中打开一个新的运行文件，并启动分子尺寸测定。在测定页面中，将样本名称输入到每个毛细管中，以及初级抗体和二级抗体的名称。在离心结束时，将板放在自动西方仪器中，然后从毛细管盒中取出盖子。离心后，不要忘记检查气泡，因为毛细管中的气泡会阻塞电泳电流，使整个圆都失效。将墨盒插入仪器内的指定位置并关闭车门。单击"开始"。当提示检测类型时，在相应的文本框中输入实验的名称，然后单击"确定"。当由激活的运行日期和运行文件中的 ID 号提示时，记下运行结束时的时间。在运行结束时，将毛细管盒丢弃到利器处置中，将板放入生物危害材料处置中。电泳分离后，检查40至43千吨抗原的免疫反应峰的运行文件，这些抗原代表磷酸化真核转化起始因子2A。如果缺少此大小的分子量峰值，请右键单击曲线下方，然后选择向峰值添加分子量，以确保记录曲线下方的大小和任意数量。在未基本组织样本中，与所有其他区域相比，单独轨道核中结合的氨基球蛋白水平要高得多。用初乳培养的肠道组织进行吸气会降低单独轨道核中结合的氨球蛋白蛋白水平，对准星体核和超视核没有影响。相反，注发增加皮层内结合的氨基球蛋白蛋白水平，条纹核，和中位预视核相对于未原始组织，这意味着在各种测试的大脑核结合氨球蛋白蛋白水平对肠道刺激的反应不同，并且尚未发现任何测试的各种核之间的串扰。真核翻译起始因子2A和磷酸化真核翻译起始因子2A水平在非原始条件下相对于其他核升高。启动后，与未基组织相比，真核翻译起始因子2A和磷酸化真核转化起始因子2A的水平相对于单独轨道的核降低。在所有其他测试的核中，注发增加真核翻译起始因子2A和磷酸化真核转化起始因子2A相对于未基质组织的水平。然而，与单独轨道核中未基质样品相比，非基质样品中磷酸化蛋白激酶受体水平较低，这强烈地表明，在真核转化起始因子2A的磷酸化中，另一个激酶也参与其中。该技术开发后，为产后开发领域的研究人员探索组织生长和分化所涉及的信号通路，以及它们是否受吮吸的影响或独立于吸吮铺平了道路。不要忘记，使用挥发性还原剂（例如二次减性剂）可能很危险，在准备本程序时应始终采取预防措施，如化学罩中的制备。

Research

JoVE Journal

Immunology and Infection

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科研

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Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

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Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

果蝇、果蝇细胞免疫应答的评估

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

具有体外转录mRNA的初级巨噬细胞的高效转染

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

通过在小鼠中吸液性淋巴血管阻止淋巴流

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to ...