我们要感谢 Kellie Breen Church 博士、Michael Kreisman 先生和 Jessica Jang 女士为收集代表性结果中显示的数据所做的贡献。这项工作得到了美国国立卫生研究院（NIH）R00 HD104994（R.B.M.）的支持。

所有程序均由科罗拉多州立大学 （#3960） 和加州大学圣地亚哥分校机构动物护理和使用委员会批准，并收集了代表性数据（S13235，PI Kellie Breen Church）。来自5只成年雌性和2只成年雄性C57 / BL6小鼠（9-16周龄）的数据在代表性数据部分中描述。如前所述，雌性小鼠在ICV注射和采血前3-4周进行卵巢切除术7。在实验之前，这些小鼠被饲养在12小时光照/ 12小时暗光循环中，并根据《实验动物护理和使用指南》自由获取饲料和水。
1. 进行开颅手术
注意：开颅手术可以在实际注射前一天或多天进行，这使得实验当天的注射过程更快。
<ol><li>如下所述准备开颅手术的材料。<ol><li>在工作台面上放置干净的台垫或悬垂材料。将异氟烷蒸发器的鼻锥粘在工作表面上（靠近实验者，但背对实验者;见 图1A）。</li>
		<li>将硅橡胶管放在无菌锋利的 18 G 针头的末端，使针尖突出 ~1 mm。将硅橡胶管放在无菌钝针的末端，使针尖突出~1毫米。 注意：每只小鼠应使用单独且无菌的预包装针头（锋利和钝）。</li>
		<li>收集电动剃须刀、碘磨砂膏、无菌纱布和酒精棉片，用于准备注射部位。</li>
	</ol></li>
	<li>如下所述，练习移动针头 2 毫米的横向和 1 毫米的尾部。<ol><li>用尺子和笔在一张纸上标记一个起点（这将是bregma），一个标记向左或向右2毫米，另一个标记在最后一个上方1毫米处（这将是注射部位;见 图1B）。</li>
		<li>练习将针头从起点移动到标记点 1 再到标记点 2（前囟到注射部位），直到确信该运动在没有导轨的情况下是可重复的。 注：此处描述的坐标在成年 C57/BL6 小鼠中有效，但其他品系或年龄组可能需要调整。</li>
	</ol></li>
	<li>如下所述准备小鼠进行开颅手术。<ol><li>将小鼠置于诱导室中，并在医用级氧气中用3%-4%异氟烷麻醉小鼠。通过练习和熟悉程序，将开颅手术的异氟烷暴露总持续时间减少到10分钟以内。 注意：异氟醚暴露对人体健康有害;在通风良好的地方使用经批准和检查的异氟烷汽化器，并清除废气。</li>
		<li>一旦脚趾反射消失，剃掉动物的头部。涂抹眼部润滑剂。</li>
		<li>将鼠标放在工作台面上，头部在鼻锥中以保持麻醉。</li>
		<li>按照供应商的指示施用镇痛剂，例如丁丙诺啡（0.6-0.8 mg/kg 小鼠体重，皮下注射）。</li>
		<li>用蘸有碘溶液的无菌纱布擦拭头部清洁注射部位（进行三次擦洗），然后用酒精擦洗垫擦拭（进行三次）。 注意：按照美国兽医协会的建议，去除毛发并清洁注射部位周围的皮肤，并使用无菌器械和针头，以防止感染。</li>
	</ol></li>
	<li>用非惯用手牢牢握住鼠标头部。将头部尽可能平放在工作台面上。</li>
	<li>找到注射部位。<ol><li>使用准备好的锋利的 18 G 针首先识别前盂;为此，将针沿着中线拖过头部皮肤，在喙尾平面上移动。想象一个等边三角形，其中两个顶点是眼睛，第三个顶点是囟的大致位置（见 图 1C）。</li>
		<li>从前乳开始，将针头向外侧移动2毫米，尾部向1毫米移动至注射部位。</li>
	</ol></li>
	<li>垂直握住针头的同时，用力将针头穿过皮肤和骨骼，直到管子与皮肤齐平。</li>
	<li>缩回针头，旋转针头，然后再次按压皮肤和骨骼。重复该过程，直到在骨头上形成一个小孔。</li>
	<li>使用钝针检查骨头上是否产生了足够的孔。目标是在骨头上开一个足够大的开口，以便钝针穿过。</li>
	<li>将鼠标从鼻锥中取出，用无菌纱布清除注射部位的任何血液，然后将鼠标放在笼子加热器上的笼子中，直到醒来。由于开颅手术的开口很小（18 G 针头），因此不需要用缝合线或伤口夹闭合该部位。 注意：这里产生的开口很小（18 G），因此许多机构认为这种手术是注射而不是手术。此外，虽然通过皮肤和骨骼向大脑开了一个口，但保持皮肤绷紧会导致孔不对齐，这可能有助于防止皮肤菌群或笼子垫料接触大脑。</li>
</ol>2. 进行注射
<ol><li>如下所述准备注射材料。<ol><li>在工作台面上放置干净的台垫或悬垂材料。将异氟烷蒸发器的鼻锥粘在工作表面上（靠近实验者，但背对实验者;见 图1A）。</li>
		<li>将一根 10 mm 长的 27 G 针头（斜角为 45°）连接到 5 μL 玻璃注射器上。将硅橡胶管放在针头上，使针尖突出 3.5 毫米;使用实验室胶带将管子固定在注射器主体上（见 图1D）。</li>
		<li>在试管中制备注射介质（药物、病毒或其他注射液）。对于本研究的代表性结果，注射无菌等渗盐水。 注意： 选择允许玻璃注射器和针头进入的试管，例如 2 mL 微量离心管。</li>
		<li>将 3 μL 注射介质吸入玻璃注射器中。首先，抽取超过所需体积的体积，然后弹出，直到注射器中残留 3 μL。</li>
		<li>收集碘磨砂膏、无菌纱布和酒精垫，用于准备注射部位。</li>
		<li>在计数模式下启动实验室计时器，并放置计时器，以便实验者在进样时可以看到它。</li>
		<li>练习像前一天一样移动针头 2 毫米的横向和 1 毫米的尾部。</li>
	</ol></li>
	<li>如下所述准备注射鼠标。<ol><li>将小鼠置于诱导室中，并用异氟醚麻醉小鼠。一旦脚趾反射消失，涂抹眼部润滑剂，然后将鼠标放在工作台面上，头部在鼻锥内。</li>
		<li>用碘和酒精各擦三片擦拭清洁注射部位。如步骤1.8所述，用18 G针（或钝针）识别注射部位。开颅手术期间形成的孔洞应该是可检测到的。 注意：如果没有提前进行开颅手术，或者如果无法检测到颅骨上的孔，则可以在此处进行开颅手术。</li>
	</ol></li>
	<li>如下所述用玻璃注射器进行注射。<ol><li>用非惯用手牢牢握住鼠标头部。将头部尽可能平放在工作台面上。</li>
		<li>将玻璃注射器的针头穿过头骨上的孔，直到放置在步骤2.1.2中制备的针头上的管子停留在小鼠的皮肤上。</li>
		<li>尽可能垂直握住注射器，注意冠状面和矢状面。在1分钟内缓慢注入培养基。</li>
		<li>注射完成后，将注射器和针头固定到位一分钟，以尽量减少回流。慢慢地将针头从鼠标头上缩回。</li>
		<li>将鼠标从鼻锥中取出，用无菌纱布（如果存在）清除注射部位的任何血液，然后将鼠标放在笼子加热器上的笼子中，直到醒来。</li>
	</ol></li>
</ol>3. 确认注射位置
<ol><li>如前所述，用盐水和4%多聚甲醛在磷酸盐缓冲盐水（PBS）中深度麻醉并灌注小鼠8。将大脑储存在4°C的PBS中的30%蔗糖中，直到大脑下沉（通常为2天）。</li>
	<li>如前所述，在低温恒温器上切割 20-50 μm 冠状切片9.切片时，观察组织块中的注射道。记录注射道是否与侧脑室明显相交。</li>
</ol>

使用徒手方法对小鼠进行脑室内注射

<ol>
	<li>Roseweir, A. 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=Discovery+of+potent+kisspeptin+antagonists+delineate+physiological+mechanisms+of+gonadotropin+regulation.">Discovery of potent kisspeptin antagonists delineate physiological mechanisms of gonadotropin regulation.</a> Journal of Neuroscience. 29 (12), 3920-3929 (2009).</li><li>Kim, J. Y., Grunke, S. D., Levites, Y., Golde, T. E., Jankowsky, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+viral+injection+of+the+neonatal+mouse+brain+for+persistent+and+widespread+neuronal+transduction.">Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction.</a> Journal of Visualized Experiments. (91), e51863 (2014).</li><li>Taylor, Z. V., Khand, B., Porgador, A., Monsonego, A., Eremenko, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+intracerebroventricular+injection+of+CD4(+)+T+cells+into+mice.">An optimized intracerebroventricular injection of CD4(+) T cells into mice.</a> STAR Protocols. 2 (3), 100725 (2021).</li><li>Russo, K. A., 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=Circadian+control+of+the+female+reproductive+axis+through+gated+responsiveness+of+the+RFRP-3+system+to+VIP+signaling.">Circadian control of the female reproductive axis through gated responsiveness of the RFRP-3 system to VIP signaling.</a> Endocrinology. 156 (7), 2608-2618 (2015).</li><li>Laursen, S. E., Belknap, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+injections+in+mice.+Some+methodological+refinements.">Intracerebroventricular injections in mice. Some methodological refinements.</a> Journal of Pharmacological Methods. 16 (4), 355-357 (1986).</li><li>Haley, T. J., McCormick, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+effects+produced+by+intracerebral+injection+of+drugs+in+the+conscious+mouse.">Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse.</a> British Journal of Pharmacology and Chemotherapy. 12 (1), 12-15 (1957).</li><li>McCosh, R. 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=Insulin-induced+hypoglycaemia+suppresses+pulsatile+luteinising+hormone+secretion+and+arcuate+Kiss1+cell+activation+in+female+mice.">Insulin-induced hypoglycaemia suppresses pulsatile luteinising hormone secretion and arcuate Kiss1 cell activation in female mice.</a> Journal of Neuroendocrinology. 31 (12), e12813 (2019).</li><li>Wu, 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=Transcardiac+perfusion+of+the+mouse+for+brain+tissue+dissection+and+fixation.">Transcardiac perfusion of the mouse for brain tissue dissection and fixation.</a> Bio-Protocol. 11 (5), e3988 (2021).</li><li>Comba, A., 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=Laser+capture+microdissection+of+glioma+subregions+for+spatial+and+molecular+characterization+of+intratumoral+heterogeneity,+oncostreams,+and+invasion.">Laser capture microdissection of glioma subregions for spatial and molecular characterization of intratumoral heterogeneity, oncostreams, and invasion.</a> Journal of Visual Experiments. (158), e60939 (2020).</li><li>Porteous, R., 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=Reformulation+of+PULSAR+for+analysis+of+pulsatile+LH+secretion+and+a+revised+model+of+estrogen-negative+feedback+in+mice.">Reformulation of PULSAR for analysis of pulsatile LH secretion and a revised model of estrogen-negative feedback in mice.</a> Endocrinology. 162 (11), (2021).</li><li>Hohmann, J. G., 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=Differential+role+of+melanocortins+in+mediating+leptin's+central+effects+on+feeding+and+reproduction.">Differential role of melanocortins in mediating leptin's central effects on feeding and reproduction.</a> American Journal of Physiology: Regulatory, Integrative, and Comparative Physiology. 278 (1), R50-R59 (2000).</li><li>Gottsch, M. 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=A+role+for+kisspeptins+in+the+regulation+of+gonadotropin+secretion+in+the+mouse.">A role for kisspeptins in the regulation of gonadotropin secretion in the mouse.</a> Endocrinology. 145 (9), 4073-4077 (2004).</li><li>Krasnow, S. M., 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=A+role+for+galanin-like+peptide+in+the+integration+of+feeding,+body+weight+regulation,+and+reproduction+in+the+mouse.">A role for galanin-like peptide in the integration of feeding, body weight regulation, and reproduction in the mouse.</a> Endocrinology. 144 (3), 813-822 (2003).</li></ol>

作者没有什么可透露的。

在这里，描述了一种在小鼠中进行ICV注射的简单有效的方法。由于该技术不需要立体定向框架，因此这种用于药物和实验剂集中递送的方法可供更多研究人员使用。此外，这种方法的通量相对较高，因为制备和注射程序可以快速执行。
由于此过程需要使用近似距离手动操作针头和玻璃注射器，因此建议新从业者在与活体动物一起工作之前花一些时间练习这些动作。此外，小鼠尸体可用于练习用针头识别囟。一旦对运动有了一定的信心，与专门的活体练习动物的经验是有帮助的。在练习过程中，注射 3 μL 印度墨水可以更容易地对注射位置进行目视评估。正确放置的注射会导致同侧脑室出现黑色染色，并且通常在第三脑室和对侧侧脑室中出现黑色染色。注射墨水是一个终末程序，不应让小鼠在注射墨水后从麻醉中恢复过来。即使是有经验的从业者也可以在注射前多次排练 2 毫米的横向和 1 毫米的尾部运动。此外，将玻璃注射器放入大脑时保持垂直也很重要。通过练习，可以预期>75%的注射剂被正确放置。在注射全部体积后将针头保持在适当的位置~1分钟有助于防止液体回流出大脑。在此期间，必须将针头保持在正确的位置而不移动。该程序的成功执行需要可靠地操作和握住针头和玻璃注射器的经验。
如果某些注射未正确放置，则有几种故障排除选项。首先，检查注射针是否笔直（不弯曲），并且针头是否牢固地固定在玻璃注射器上。接下来，如果错过的注射位置各不相同，则需要进一步练习操作和握住玻璃注射器。在注射过程中，让额外的人来发现注射器是否垂直握住也会有所帮助。用针头在完整的皮肤上发现前囟也需要练习。强烈建议在进行注射之前花点时间自信地识别这个标志。如果进样始终放置在不正确的位置，则需要更改进样坐标。这里描述的坐标（±2 mm横向，1 mm尾部到前囟）对成年C57 / BL6小鼠有效，但这些坐标可能必须针对其他菌株或年龄组进行调整。
尽管这种技术在集中快速交付代理方面非常有效，但存在一些局限性。由于注射器的柱塞是用手移动的，因此注射速率可以是可变的。建议相对缓慢地注射，以避免心室系统中压力的潜在脱靶效应。在该方案中，推荐注射体积为3μL;然而，其他实验室也使用了更大的进样体积（5 μL）11。根据测量的结果，可能需要调整注射量和速率。例如，幼年小鼠可能只能耐受较小的注射量。因此，建议进行初步实验来测试车辆效应。另一个局限性是，正确注射位置的确认仅限于在收集组织后对注射道进行某种主观的评估。在ICV注射数小时后收集的新鲜冷冻和多聚甲醛灌注组织中均可见注射道（见代表性结果中的图像）。如果多次注射，可能无法从道痕中看出每次注射是否成功放置。
这种徒手注射 ICV 的方法需要麻醉。通过练习，可以快速进行注射，导致短暂（3-5 分钟）暴露于麻醉剂。通过在实验日之前剃掉小鼠的头部并进行开颅手术（用锋利的针刺穿头骨），可以减少实验期间的麻醉时间。小鼠通常会在几分钟内从这种短暂的麻醉暴露中完全恢复。麻醉后立即收集尾尖血样可能具有挑战性，因此可以在注射后暂停采样~30分钟，以尽量减少小鼠的压力，尽管这不是绝对必要的。其他实验室也成功地测量了使用这种徒手 ICV技术施用药物后 30 分钟LH分泌（一次性样本）的变化1,12。在注射 ICV 后，还检测到 24 小时内运动和摄入行为的变化13，因此可以进行长期监测。建议进行初步测试以确定取样的难易程度以及麻醉对感兴趣结局的潜在影响。
总之，这里描述的徒手ICV注射技术是一种将实验剂输送到大脑的简单而强大的方法。具体优点是该方法快速且技术简单，并且可以实现高成功放置注射率。此外，由于该技术只需要短暂暴露于麻醉，因此可以与各种其他技术相结合，例如频繁采血、神经回路操作或 体内 记录以研究神经内分泌过程。

将实验药物（如药物1、病毒2 或细胞3）输送到大脑通常是神经内分泌研究所必需的。如果该药剂不容易穿过血脑屏障，或者实验目标是专门测试药剂的中枢作用，那么有一种可靠的方法将注射剂输送到大脑中是很重要的。此外，注射到脑室内 （ICV） 空间提供了将药物广泛分布在大脑中的机会，并提供了较大的靶区，从而增加了成功注射的可能性2. 
进行 ICV 注射的常用方法是放置永久性留置插管。在这种方法中，需要立体定位框架来定位市售或定制的套管，因为套管被粘合或胶合到位。通常，在康复后，通过套管给予超生理剂量的血管紧张素 II，如果立即观察到饮酒行为，则认为套管放置正确4.这种方法具有许多优点，包括能够进行长期输注和多次注射同一只动物的能力;此外，如果使用血管紧张素II，则可以在施用实验化合物之前确认正确的放置。然而，放置永久性插管存在一些局限性，包括对昂贵设备（立体定位框架）的要求、放置后插管损坏的可能性（例如，小鼠可以咀嚼笼子伴侣的插管）以及永久性插管周围感染的可能性。可以使用立体定位框架3 进行单次 ICV 注射，这虽然有效，但需要大量暴露于麻醉，因此可能会掩盖治疗的一些急性生理和行为影响。此外，将小鼠放置在立体定位框架中需要大量训练才能实现稳定放置并防止耳道破裂。
在这里，描述了一种在小鼠中进行徒手注射的既定方法。这种方法基于以前的报告5,6。这种技术的优点是简单、快速，不需要立体定位框架等专用设备。如下所述，该过程涉及操纵玻璃注射器相对于小鼠头部的标志进行注射，这可以快速完成，因此在实验当天只需要几分钟的气体麻醉。

<table><tbody><tr><td>18-gauge blunt needles</td><td>SAI Infusion</td><td>B18-150</td><td></td></tr><tr><td>18-gauge needles</td><td>BD Medical</td><td>305195</td><td></td></tr><tr><td>Alcohol pads</td><td>Fisher Scientific</td><td>22-363-750</td><td></td></tr><tr><td>Bench pad</td><td>Fisher Scientific</td><td>14-206-62AC22</td><td></td></tr><tr><td>Betadine solution</td><td>Fisher Scientific</td><td>NC1696484</td><td></td></tr><tr><td>Buprenorphine</td><td>Patterson Vet Supply</td><td>07-892-5235</td><td>Controlled substance</td></tr><tr><td>Eyelube</td><td>Fisher Scientific</td><td>50-218-8442</td><td></td></tr><tr><td>Glass syringe</td><td>Hamilton</td><td>7634-01</td><td></td></tr><tr><td>Injection needle</td><td>Hamilton</td><td>7803-01</td><td>27 gauge, Small Hub RN needle, point style: 4, Needle length: 10cm, Angle: 45</td></tr><tr><td>Isoflurane&amp;nbsp;&amp;nbsp;</td><td>Patterson Vet Supply</td><td>07-893-8441</td><td></td></tr><tr><td>Isoflurane vaporizer</td><td>Vet Equip</td><td>V-10</td><td></td></tr><tr><td>Laboratory Tape</td><td>VWR</td><td>89098-128</td><td></td></tr><tr><td>Medical grade oxygen</td><td>Airgas</td><td>OX USPEA</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Millipore-Sigma</td><td>8.18715.1000</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Fisher Scientific</td><td>J67802.K2</td><td></td></tr><tr><td>PulsaR Software</td><td>Open source, University of Otago</td><td></td><td>See ref 9</td></tr><tr><td>Ruler</td><td>Fisher Scientific</td><td>12-00-152</td><td></td></tr><tr><td>Silastic tubing (0.040&quot; I.D.)</td><td>DOW</td><td>508-005</td><td></td></tr><tr><td>Silastic tubing (0.078&quot; I.D.)</td><td>DOW</td><td>508-009</td><td></td></tr><tr><td>Sterile saline</td><td>VWR</td><td>101320-574</td><td></td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Fisher Scientific</td><td>S5-500</td><td></td></tr></tbody></table>

free-hand intracerebroventricular injections in mice

神经内分泌系统的研究通常需要将药物、病毒或其他实验剂直接输送到小鼠的大脑中。脑室内 （ICV） 注射允许实验药物在整个大脑中广泛递送（特别是在心室附近的结构中）。在这里，描述了在成年小鼠中进行徒手ICV注射的方法。通过在小鼠头部使用视觉和触觉标志，可以快速可靠地向侧脑室注射。注射是用实验者手中的玻璃注射器进行的，并放置在与地标大致距离的地方。因此，该技术不需要立体定位框架。此外，该技术只需要短暂的异氟醚麻醉，这允许随后评估清醒、行为自由的小鼠的小鼠行为和/或生理学。徒手ICV注射是将实验剂有效输送到活体小鼠大脑中的强大工具，可以与其他技术相结合，如频繁的血液采样、神经回路操作或 体内 记录，以研究神经内分泌过程。

成功执行后，该技术允许将实验剂快速输送到心室系统中。 图2A显示了接受ICV注射3μL无菌等渗盐水（许多药理学化合物的载体）的卵巢切除小鼠的促黄体激素（LH）脉冲曲线。该示例表明，短暂暴露于气体麻醉和单独向心室系统注射 3 μL 液体不会改变搏动性 LH 分泌。ICV注射后3 h，对动物实施安乐死，收集新鲜冷冻的神经组织，在低温恒温器上切割;注射道与侧脑室明显相交。在灌注固定剂（10 mL 肝素化盐水，然后在磷酸盐缓冲液中加入 20 mL 4% 多聚甲醛）的小鼠神经组织中也可见注射道。正确放置进样的示例如图 2B所示。 图2C显示了两个错误放置进样的示例。正确（左）和错误放置印度墨水的示例如图 2D 所示。印度墨水的注入对于练习这种技术很有用，因为它可以清晰地可视化注射位置。在正确放置的注射中可以看到染料流入侧脑室和第三脑室（图2D，左）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65324/65324fig01.jpg"> 图 1：描绘设备准备情况的图像和 徒手 ICV 注射的注射坐标。 （A） 徒手ICV注射工作站的配置。（B） 练习在 3-4 个月大的雌性 C57/BL6 小鼠中放置 ICV 注射所需的运动的方案。（C）成年小鼠囟的大致位置。（D） 放置管子以露出注射针尖 3.5 毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65324/65324fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65324/65324fig02.jpg"> 图 2：与徒手 ICV 注射相关的代表性黄体生成素和组织学数据。 （A） ICV 注射前后的 LH 脉冲曲线。带有黑色实心填充的数据点表示使用 PULSAR10 的重新配方确定的脉冲，该参考中建议的参数用于 OVX 小鼠，检测水平为 0.32 ng/mL。（B） 正确放置 ICV 注射的小鼠大脑冠状切片的照片。（C） ICV注射位置不正确的小鼠大脑冠状部分的照片：脑室外侧的注射道（左）和脑室背侧的注射道（右）。（D） 正确放置 ICV 注射印度墨水（左）和错误放置 ICV 注射印度墨水（右）的小鼠大脑冠状切片的照片。注：图 B 和 C 描绘了注射生理盐水3小时后收集的多聚甲醛灌注脑;图 D 描绘了注射印度墨水2分钟后在干冰上收集的新鲜大脑。比例尺 = 1 mm <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65324/65324fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Insights, Innovations, and Future Avenues in Reproductive Neurocontrol at JoVE.com

作者聚焦：生殖神经控制的见解、创新和未来途径 

在这里，描述了一种使用徒手方法（即没有立体定位装置）在小鼠中进行脑室内注射的简单快速的方法。

小鼠徒手脑室内注射

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. ...

我们对生殖功能的神经控制感兴趣，特别是大脑如何调节控制性腺的激素的释放，无论是在健康条件下还是在应对压力时。这种技术的主要优点是相对简单和快速。将药物输送到侧脑室是在测试信号通路以支持更详细的后续实验时广撒网的好方法。关于生殖的神经内分泌控制还有很多东西需要了解，包括促黄体生成素脉冲的分子机制、应激期间调节脉冲频率的机制以及排卵前 LH 激增的产生。未来，我们对将不同的发现整合到模型中特别感兴趣。

free-hand-intracerebroventricular-injections-in-mice

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Free-Hand Intracerebroventricular Injections in Mice

10.3K 次观看.  Colorado State University. 我们对生殖功能的神经控制感兴趣，特别是大脑如何调节控制性腺的激素的释放，无论是在健康条件下还是在应对压力时。这种技术的主要优点是相对简单和快速。将药物输送到侧脑室是在测试信号通路以支持更详细的后续实验时广撒网的好方法。关于生殖的神经内分泌控制还有很多东西需要了解，包括促黄体生成素脉冲的分子机制、应激期间调节脉冲频率的机制以及?...

视频: 作者聚焦：生殖神经控制的见解、创新和未来途径 

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

脑浸润白细胞的分离

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

科研

免疫与感染

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

交付的治疗药物通过侧脑室（ICV）和静脉（IV）注射液在小鼠

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

医学

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

自体血液的注入模型小鼠自发性脑出血

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

神经科学

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

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 ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

啮齿动物脑 Microinjections 的个性化针

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

在新生小鼠静脉注射

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

生物学

Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have revealed that gut microbiota-derived metabolites modulate brain-mediated physiological functions through intricate gut-brain pathways in the host. Short-chain fatty acids (SCFAs) are the major bacteria-derived metabolites produced during dietary fiber fermentation by the gut microbiome. Secreted SCFAs from the gut can act at multiple sites in the periphery, affecting the immune, endocrine, and neural responses due to the vast distribution of SCFAs receptors. Therefore, it is challenging to differentiate the central and peripheral effects of SCFAs through oral and intraperitoneal administration of SCFAs. This paper presents a video-based method to interrogate the functional role of SCFAs in the brain via a guide cannula in freely moving mice. The amount and type of SCFAs in the brain can be adjusted by controlling the infusion volume and rate. This method can provide scientists with a way to appreciate the role of gut-derived metabolites in the brain.

Gut-derived microbial metabolites have multifaceted effects leading to complex behavior in animals. We aim to provide a step-by-step method to delineate the effects of gut-derived microbial metabolites in the brain via intracerebroventricular delivery via a guide cannula.

自由移动小鼠中肠道来源的微生物代谢物的脑室内递送

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have ...

Handling-Induced Seizures in a Mouse Model

Source: Batot, G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63673">A Model for Epilepsy of Infectious Etiology using Theiler&#39;s Murine Encephalomyelitis Virus.</a>&#160;J. Vis. Exp. (2022).
This video demonstrates the induction of handling-induced seizures in a mouse infected with Theiler&#39;s Murine Encephalomyelitis Virus (TMEV). The infected mouse, which has hyperexcitable neurons, is exposed to external physical stimuli. This exposure causes stress, leading to abnormal electrical activity in the neurons, which results in seizures.

Source: Batot, G., et al. A Model for Epilepsy of Infectious Etiology using Theiler&#39;s Murine Encephalomyelitis Virus.&#160;J. Vis. Exp. (2022).
This ...

JoVE Encyclopedia of Experiments

Neuropathology

Epilepsy and Seizure Disorders

Glioblastoma Induction Using SB Mediated Transposition: A Technique for Generating Novel Mouse Model Using Transposon Mediated Integration of SB Plasmid into Neonatal Mouse Brain

Source: Calinescu, A. A. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/52443" target="_blank">Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma.</a> J. Vis. Exp. (2015)
This video describes an efficient technique to induce glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate, and generate tumors with histopathological characteristics of human GBM.

使用 SB 介导的转座诱导胶质母细胞瘤:一种使用转座子介导的 SB 质粒整合到新生小鼠脑中生成新型小鼠模型的技术

Source: Calinescu, A. A. et al. Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of ...