Name: stereotaxic surgical approach to microinject the caudal brainstem and upper cervical spinal cord via the cisterna magna in mice
Uploaded: 2022-01-21T14:00:00
Description: Watch this Scientific Journal Video about Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice at JoVE.com

这项工作得到了R01 NS079623，P01 HL149630和P01 HL095491的支持。

作者声明，该协议遵循Beth Israel Deaconess医疗中心机构动物护理和使用委员会的指导方针。
1. 手术器械和立体定位框架的准备
注意：手术是在无菌条件下进行的。使用无菌尖端技术保持无菌状态。
<ol><li>在立体定位框架上安装带有微量移液管或注射器的立体定位臂，其中装有所选注射剂（腺相关病毒（AAV）或常规示踪剂），并准备鼠标适配?...

<ol>
	<li>JoVE. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+Stereotaxic+Surgery.">Rodent Stereotaxic Surgery.</a> JoVE Science Education Database. , (2021).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2001).</li><li>Lein, E. 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=Genome-wide+atlas+of+gene+expression+in+the+adult+mouse+brain.">Genome-wide atlas of gene expression in the adult mouse brain.</a> Nature. 445 (7124), 168-176 (2007).</li><li>Rangarajan, J. 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=Image-based+in+vivo+assessment+of+targeting+accuracy+of+stereotactic+brain+surgery+in+experimental+rodent+models.">Image-based in vivo assessment of targeting accuracy of stereotactic brain surgery in experimental rodent models.</a> Scientific Reports. 6 (1), 38058 (2016).</li><li>Blasiak, T., Czubak, W., Ignaciak, A., Lewandowski, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+detection+of+the+bregma+point+on+the+rat+skull.">A new approach to detection of the bregma point on the rat skull.</a> Journal of Neuroscience Methods. 185 (2), 199-203 (2010).</li><li>Popesko, P., Rajtova, V., Horak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Colour Atlas of the Anatomy of Small Laboratory Animals, Volume 2: Rat, Mouse and Golden Hamster. 2, (1992).</li><li>Allen Mouse Brain Atlas. Allen Institute for Brain Science Available from: <a target="_blank" href="https://mouse.brain-map.org/experiment/thumbnails/100042147?image_type=atlas">https://mouse.brain-map.org/experiment/thumbnails/100042147?image_type=atlas</a> (2004)</li><li>Vanderhorst, V. G. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleus+retroambiguus-spinal+pathway+in+the+mouse:+Localization,+gender+differences,+and+effects+of+estrogen+treatment.">Nucleus retroambiguus-spinal pathway in the mouse: Localization, gender differences, and effects of estrogen treatment.</a> The Journal of Comparative Neurology. 488 (2), 180-200 (2005).</li><li>Yokota, S., Kaur, S., VanderHorst, V. G., Saper, C. B., Chamberlin, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory-related+outputs+of+glutamatergic,+hypercapnia-responsive+parabrachial+neurons+in+mice.">Respiratory-related outputs of glutamatergic, hypercapnia-responsive parabrachial neurons in mice.</a> Journal of Comparative Neurology. 523 (6), 907-920 (2015).</li><li>Anselmi, C., 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=Ultrasonographic+anatomy+of+the+atlanto-occipital+region+and+ultrasound-guided+cerebrospinal+fluid+collection+in+rabbits+(Oryctolagus+cuniculus).">Ultrasonographic anatomy of the atlanto-occipital region and ultrasound-guided cerebrospinal fluid collection in rabbits (Oryctolagus cuniculus).</a> Veterinary Radiology &amp; Ultrasound. 59 (2), 188-197 (2018).</li><li>Herbert, H., Moga, M. M., Saper, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connections+of+the+parabrachial+nucleus+with+the+nucleus+of+the+solitary+tract+and+the+medullary+reticular+formation+in+the+rat.">Connections of the parabrachial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat.</a> The Journal of Comparative Neurology. 293 (4), 540-580 (1990).</li><li>Vanderhorst, V. G., Holstege, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caudal+medullary+pathways+to+lumbosacral+motoneuronal+cell+groups+in+the+cat:+evidence+for+direct+projections+possibly+representing+the+final+common+pathway+for+lordosis.">Caudal medullary pathways to lumbosacral motoneuronal cell groups in the cat: evidence for direct projections possibly representing the final common pathway for lordosis.</a> The Journal of Comparative Neurology. 359 (3), 457-475 (1995).</li><li>Vanderhorst, V. G., Terasawa, E., Ralston, H. J., Holstege, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+projections+from+the+nucleus+retroambiguus+to+motoneurons+supplying+the+abdominal+wall,+axial,+hindlimb,+and+pelvic+floor+muscles+in+the+female+rhesus+monkey.">Monosynaptic projections from the nucleus retroambiguus to motoneurons supplying the abdominal wall, axial, hindlimb, and pelvic floor muscles in the female rhesus monkey.</a> The Journal of Comparative Neurology. 424 (2), 233-250 (2000).</li><li>Wall, N. R., Wickersham, I. R., Cetin, A., De La Parra, M., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+circuit+tracing+in+vivo+through+Cre-dependent+targeting+and+complementation+of+modified+rabies+virus.">Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (50), 21848-21853 (2010).</li><li>Krashes, M. 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=Rapid,+reversible+activation+of+AgRP+neurons+drives+feeding+behavior+in+mice.">Rapid, reversible activation of AgRP neurons drives feeding behavior in mice.</a> The Journal of Clinical Investigation. 121 (4), 1424-1428 (2011).</li><li>Ganchrow, 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=Nucleus+of+the+solitary+tract+in+the+C57BL/6J+mouse:+Subnuclear+parcellation,+chorda+tympani+nerve+projections,+and+brainstem+connections.">Nucleus of the solitary tract in the C57BL/6J mouse: Subnuclear parcellation, chorda tympani nerve projections, and brainstem connections.</a> The Journal of Comparative Neurology. 522 (7), 1565-1596 (2014).</li><li>Ung, K., Arenkiel, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber-optic+implantation+for+chronic+optogenetic+stimulation+of+brain+tissue.">Fiber-optic implantation for chronic optogenetic stimulation of brain tissue.</a> Journal of Visualized Experiments: JoVE. (68), e50004 (2012).</li></ol>

标准立体定向手术通常依靠颅骨特征来计算中枢神经系统1中靶位点的坐标。然后 通过 钻入颅骨1的毛刺孔进入目标部位。这种方法对于尾部脑干来说并不理想，因为靶点位于前后平面和背腹平面2中远离颅骨标志点， 并且颅骨和上覆肌肉的解剖结构使得进入具有挑战性6 （图1Bi）。我们的研究描?...

针对小鼠1 的大脑部位的标准立体定位手术通常涉及使用一组耳杆和嘴条固定颅骨。然后根据参考图集2，3和颅骨特征估计坐标，即前颌骨（额骨和顶骨缝合在一起的点）或λ（顶骨和枕骨缝合在一起的点; 图1A、B）。通过进入估计目标上方颅骨的毛刺孔，然后可以到达目标区域，用于传递显微注射或带有插管或光纤的仪器。由于这些缝合线的解剖结构的变化以及bregma或lambda4，5定位的错误，零点相对于大脑的位置因动物而异。虽然由于这种变异性而导致的小目标误差对于大型或附近的目标来说不是问题，但对于远离前后或背阴平面零点的较小感兴趣区域和/或研究由于年龄，菌株和/或性别而不同大小的动物时，它们的影响更大。延髓和上颈髓还有几个独特的挑战。首先，由于小脑的位置和形状，前后坐标的微小变化与背腹坐标相对于硬脑膜的显着变化有关（图1Bi）2，6，7。其次，上颈带不包含在颅骨2内。第三，枕骨和颈部肌肉2 的上覆层的倾斜位置使得标准立体定位方法对于位于脑干和脊髓之间过渡附近的结构更具挑战性（图1Bi）。最后，尾部脑干和颈带中许多感兴趣的靶标是小2，需要精确和可重复的注射8，9。
通过大蓄水池的另一种方法规避了这些问题。大水箱是从枕骨延伸到图谱的大空间（图1A，即第二椎骨）10。它充满脑脊液，并由硬脑膜10覆盖。枕骨和图谱之间的这个空间在头部前屈时打开。它可以通过在长头炎肌肉的上覆成对的腹部之间导航来访问，暴露尾部脑干的背表面。然后，如果感兴趣区域位于背表面附近，则可以根据这些区域本身的地标来定位这些区域;或者通过使用obex，即中央管通向IV心室的点，作为坐标到达更深结构的零点。这种方法已成功用于多种物种，包括大鼠11，猫12，小鼠8，9和非人灵长类动物13 ，以靶向腹侧呼吸组，髓质内侧网状结构，孤束核，区域后或舌下核。然而，这种方法并未得到广泛使用，因为与标准立体定位方法相比，它需要解剖学知识，专门的工具包和更高级的手术技能。
在这里，我们描述了一种循序渐进的手术方法， 通过 大水箱到达脑干和上颈髓，可视化标志点，设置零点（图2），并估计和优化目标坐标，以便将显微注射物立体定向传递到感兴趣的离散脑干和脊髓区域（图3）。然后，我们讨论与此方法相关的优缺点。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alcohol pad</td>
			<td>Med-Vet International</td>
			<td>SKU: MDS090735Z</td>
			<td>skin preparation for the prevention of surgical site infection</td>
		</tr>
		<tr>
			<td>Angled forceps, Dumont #5/45</td>
			<td>FST</td>
			<td>11251-35</td>
			<td>only to grab dura</td>
		</tr>
		<tr>
			<td>Betadine pad</td>
			<td>Med-Vet International</td>
			<td>SKU:PVP-PAD</td>
			<td>skin preparation for the prevention of surgical site infection</td>
		</tr>
		<tr>
			<td>Cholera toxin subunit-b, Alexa Fluor 488/594 conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>488: C34775, 594: C22842</td>
			<td>Fluorescent tracer</td>
		</tr>
		<tr>
			<td>Clippers</td>
			<td>Wahl</td>
			<td>Model MC3, 28915-10</td>
			<td>for shaving fur at surgical site</td>
		</tr>
		<tr>
			<td>Electrode holder with corner clamp</td>
			<td>Kopf</td>
			<td>1770</td>
			<td>to hold glass pipette</td>
		</tr>
		<tr>
			<td>Flowmeter</td>
			<td>Gilmont instruments</td>
			<td>model # 65 MM</td>
			<td>to regulate flow of isoflurane and oxygen to mouse on the surgical plane</td>
		</tr>
		<tr>
			<td>Fluorescent microspheres, polystyrene</td>
			<td>Thermo Fisher Scientific</td>
			<td>F13080</td>
			<td>Fluorescent tracer</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Stoelting</td>
			<td>53800M</td>
			<td>thermoregulation</td>
		</tr>
		<tr>
			<td>Induction chamber with port hook up kit</td>
			<td>Midmark Inc</td>
			<td>93805107 92800131</td>
			<td>chamber providing initial anasthesia</td>
		</tr>
		<tr>
			<td>Insulin Syringe</td>
			<td>Exelint International</td>
			<td>26028</td>
			<td>to administer saline and analgesic</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Med-Vet International</td>
			<td>SKU:RXISO-250</td>
			<td>inhalant anesthetic</td>
		</tr>
		<tr>
			<td>Isoflurane Matrix VIP 3000 vaporizer</td>
			<td>Midmark Inc</td>
			<td>91305430</td>
			<td>apparatus for inhalant anesthetic delivery</td>
		</tr>
		<tr>
			<td>Laminectomy forceps, Dumont #2</td>
			<td>FST</td>
			<td>11223-20</td>
			<td>only to clean dura</td>
		</tr>
		<tr>
			<td>Medical air, compressed</td>
			<td>Linde</td>
			<td>UN 1002</td>
			<td>used with stimulator &#38; PicoPump for providing air for precision solution injection</td>
		</tr>
		<tr>
			<td>Meloxicam SR</td>
			<td>Zoo Pharm LLC</td>
			<td>Lot # MSR2-211201</td>
			<td>analgesic</td>
		</tr>
		<tr>
			<td>Microhematocrit borosilicate glass pre calibrated capillary tube</td>
			<td>Globe Scientific Inc</td>
			<td>51628</td>
			<td>for transfection of material to designated co-ordinates</td>
		</tr>
		<tr>
			<td>Mouse adaptor</td>
			<td>Stoelting</td>
			<td>0051625</td>
			<td>&#160;adapting rat stereotaxic frame for mouse surgery</td>
		</tr>
		<tr>
			<td>Needle holder, Student Halsted- Mosquito Hemostats</td>
			<td>FST</td>
			<td>91308-12</td>
			<td>for suturing</td>
		</tr>
		<tr>
			<td>Oxygen regulator</td>
			<td>Life Support Products</td>
			<td>S/N 909328, lot 092109</td>
			<td>regulate oxygen levels from oxygen tank</td>
		</tr>
		<tr>
			<td>Oxygen tank, compressed</td>
			<td>Linde</td>
			<td>USP UN 1072</td>
			<td>provided along with isoflurane anasthesia</td>
		</tr>
		<tr>
			<td>Plastic card</td>
			<td>not applicable</td>
			<td>not applicable</td>
			<td>any firm plastic card, cut to fit the stereotactic frame (e.g. ID card)</td>
		</tr>
		<tr>
			<td>Pneumatic PicoPump ( or similar)</td>
			<td>World Precision Instruments (WPI)</td>
			<td>SYS-PV820</td>
			<td>For precision solution injection</td>
		</tr>
		<tr>
			<td>Saline, sterile</td>
			<td>Mountainside Medical Equipment</td>
			<td>H04888-10</td>
			<td>to replace body fluids lost during surgery</td>
		</tr>
		<tr>
			<td>Scalpel handle, #3</td>
			<td>FST</td>
			<td>10003-12</td>
			<td>to hold scalpel</td>
		</tr>
		<tr>
			<td>Scissors, Wagner</td>
			<td>FST</td>
			<td>14070-12</td>
			<td>to cut polypropylene suture</td>
		</tr>
		<tr>
			<td>Spring scissors, Vannas 2.5mm with accompanying box</td>
			<td>FST</td>
			<td>15002-08</td>
			<td>scissors only to open dura, box to elevate body</td>
		</tr>
		<tr>
			<td>Stereotactic micromanipulator</td>
			<td>Kopf</td>
			<td>1760-61</td>
			<td>attached to electrode holder to adjust position based on co-ordinates</td>
		</tr>
		<tr>
			<td>Stereotactic &#39;U&#39; frame assembly and intracellular base plate</td>
			<td>Kopf</td>
			<td>1730-B, 1711</td>
			<td>frame for surgery</td>
		</tr>
		<tr>
			<td>Sterile cotton tipped applicators</td>
			<td>Puritan</td>
			<td>25-806 10WC</td>
			<td>absorbing blood from surgical field</td>
		</tr>
		<tr>
			<td>Sterile non-fenestrated drapes</td>
			<td>Henry Schein</td>
			<td>9004686</td>
			<td>for sterile surgical field</td>
		</tr>
		<tr>
			<td>Sterile opthalmic ointment</td>
			<td>Puralube</td>
			<td>P1490</td>
			<td>ocular lubricant</td>
		</tr>
		<tr>
			<td>Stimulator &#38; Tubing</td>
			<td>Grass Medical Instruments</td>
			<td>S44</td>
			<td>to provide controlled presurred air for precision solution injection</td>
		</tr>
		<tr>
			<td>Surgical Blade #10</td>
			<td>Med-Vet International</td>
			<td>SKU: 10SS</td>
			<td>for skin incision</td>
		</tr>
		<tr>
			<td>Surgical forceps, Extra fine Graefe</td>
			<td>FST</td>
			<td>11153-10</td>
			<td>to hold skin</td>
		</tr>
		<tr>
			<td>Surgical gloves</td>
			<td>Med-Vet International</td>
			<td>MSG2280Z</td>
			<td>for asceptic surgery</td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Leica</td>
			<td>Model M320/ F12</td>
			<td>for 5X-40X magnification of surgical site</td>
		</tr>
		<tr>
			<td>Suture 5-0 polypropylene</td>
			<td>Oasis</td>
			<td>MV-8661</td>
			<td>to close the skin</td>
		</tr>
		<tr>
			<td>Tegaderm</td>
			<td>3M</td>
			<td>3M ID 70200749250</td>
			<td>provides sterile barrier</td>
		</tr>
		<tr>
			<td>Universal Clamp and stand post</td>
			<td>Kopf</td>
			<td>1725</td>
			<td>attached to stereotactic U frame and intracellular base plate</td>
		</tr>
		<tr>
			<td>Wound hook with hartman hemostats</td>
			<td>FST</td>
			<td>18200-09, 13003-10</td>
			<td>to separate muscles and provide surgical window</td>
		</tr>
	</tbody>
</table>

stereotaxic surgical approach to microinject the caudal brainstem and upper cervical spinal cord via the cisterna magna in mice

靶向小鼠大脑部位的立体定位手术通常由颅骨标志物引导。然后 通过 钻入颅骨的毛刺孔获得通道。由于特定的解剖学挑战，这种标准方法对于尾部脑干和上颈带的靶标可能具有挑战性，因为这些部位远离颅骨标志物，导致不精确。在这里，我们概述了一种 通过 大蓄水池的替代立体定位方法，该方法已用于靶向尾脑干和上颈髓中感兴趣的离散区域。大池从枕骨延伸到图谱（即第二椎骨），充满脑脊液，并被硬脑膜覆盖。这种方法为选择中枢神经系统（CNS）结构提供了一条可重复的途径，这些结构由于解剖学障碍而难以到达。此外，它允许直接可视化靠近目标部位的脑干特征，提高将小注射量输送到尾部脑干和上颈髓中受限的感兴趣区域时的准确性。最后，这种方法提供了避免小脑的机会，小脑对运动和感觉运动研究很重要。

大水池方法可以靶向尾部脑干和上颈带结构，否则这些结构很难通过标准立体定位方法到达或容易靶向不一致。到达大水池的手术需要切口皮肤，薄薄的梯形肌层和硬脑膜的开口，因此小鼠耐受性良好。当针对多个（纵向分散或双侧）地点时，它特别有效且侵入性更小，因为它不需要像标准立体定位方法那样钻多个毛刺孔。在小鼠中，我们使用大水池方法常规靶向结构，例如<...

Watch this Scientific Journal Video about Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice at JoVE.com

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

针对小鼠大脑部位的立体定向手术通常涉及通过颅骨进入，并由颅骨特征引导。在这里，我们概述了一种替代立体定位方法， 通过 大水箱靶向尾部脑干和上颈椎脊髓，该方法依赖于脑干特征的直接可视化。

立体定位手术方法 ，通过 大水池在小鼠中显微注射尾部脑干和上颈椎脊髓

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. ...

该协议为进入尾脑干和上颈髓中感兴趣区域的可重复途径提供了分步指南。当向尾脑干和上颈髓中受限的感兴趣区域提供小注射量时，该技术可提高精度。这种技术已经并且可以应用于其他动物模型。Veronique VanderHorst博士，神经病学副教授和VanderHorst实验室的首席研究员，将演示这一程序。确保氧气流向鼻锥。将鼠标移动到立体定位框架，并将鼻子放在柔性鼻锥中。仅使用耳条将鼠标放入立体定位框架中。将润滑剂涂抹在眼睛上。通过手动引导鼻子将鼠标头部向90度角弯曲。要固定此位置，请在鼠标适配器的耳杆支柱之间放置一个塑料屏障，该支柱与支柱平行，头骨的平坦部分作为参考。将加热垫放在鼠标下方，并通过用小盒子抬高身体，确保颈部和身体的其他部位与桌子平行放置。在身体下方放置一个垂坠物。注射每公斤四毫克美洛昔康的单剂量，皮下缓慢释放，体积为每克体重两微升。首先用70%酒精制备垫清洁手术切口部位，然后用甜菜碱制备垫清洁，然后用酒精制备垫再次清洁，让它干燥。消毒双手并戴上无菌手套，然后在手术部位放置窗帘。通过捏脚趾或检查角膜反射，确保对鼠标进行适当的麻醉。减少异氟醚以将水平维持在2.0。用手术10厘米的切口从枕骨边缘到肩膀做一个1到1.2厘米的切口，一个平滑的运动。小心地在斜方肌的中线裂缝中切开一个切口，露出成对的长头肌。将两个牵开器钩放在成对的长头炎肌肉之间，一个朝左，另一个朝向右。使用钝性椎板切除术镊子分离成对的长头炎肌肉的左腹和右腹，从枕骨开始，其中中线很容易看到。引导钝镊子穿过中线的枕骨，直到它与蓄水池硬脑膜相遇的地方，然后继续穿过硬脑膜到图集。重新定位牵开器，并通过重新定位止血器来调整张力。使用钝性椎板切除术镊子在中线进一步分离肌肉，以获得脑干和小脑的良好视野。根据需要重复上述步骤，直到小脑和脑干出现在硬脑膜下方。使用钝性椎板切除术镊子，通过将镊子从中线向横向移动，直到有清晰的脑干视图，从而清除结缔组织小链的硬脑膜，从而产生更多的横向空间。通过开放的硬脑膜查看脑干的背表面，并附上详细的地标。使用倾斜的镊子抓住从枕骨延伸到图集的硬脑膜，然后使用弹簧剪刀在硬脑膜上做一个大约0.5至1.5毫米的小开口。一旦硬脑膜打开，用无菌Q头排出多余的脑脊液。obex是中央管通向第四脑室的点，是标准的前后零点和中外侧零点。使用立体定位臂将移液器或注射器定位到目标。将背侧臂降低到背侧表面，背侧表面由背侧零点形成。然后将移液器放到脑干上并注射溶液。注射后将针头留在原位一到五分钟，以避免针头轨道，当使用体积在3到50纳升之间时。然后使用立体定位臂提起移液器或注射器，并对多个目标重复此操作。小心地从手术场上取下钩子。成对的长头炎肌肉将落回中立位置，完全覆盖大水池。不要在中线关闭斜方肌和硬脑膜，因为它们太脆弱而无法容纳缝合线。用3个尼龙或聚丙烯缝合线闭合皮肤。大水箱入路可以靶向尾部脑干和上颈带结构，否则这些结构很难通过标准立体定位方法到达或容易靶向不一致。在小鼠中，诸如舌下核，腹侧呼吸组和尾部脑干中的相邻网状结构已使用大水箱方法常规靶向，如图所示，用于舌下核和腹内侧髓质。为了确定大水池进近与标准进近的准确性，测量了腹侧和背侧区域的前后平面，中外侧和背侧平面中预期和实际目标位点之间的距离。结果显示，与标准方法相比，前后平面、中外平面和背阴平面的误差明显更小，突出了大水池进近对这些目标的更高精度。在尝试此方案时，确保头部的前屈和身体的抬高按所述进行至关重要。接下来，在操纵肌肉或硬脑膜之前识别关键标志非常重要。如果这些地标无法识别或丢失，则保持方向并按预期执行程序将具有挑战性。该技术有助于解决与尾脑干和上颈髓内功能解剖组织有关的概念性问题。

Research

JoVE Journal

Neuroscience

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

脊髓电二：外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

科研

神经科学

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Spinal Cord Transection in the Larval Zebrafish

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Diffusion Imaging in the Rat Cervical Spinal Cord

在大鼠颈脊髓弥散成像

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Cannula Implantation into the Cisterna Magna of Rodents

将套管植入啮齿动物的麦格纳池中

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

头脊髓和薄片体外制备心律失常活性

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

A Revised Surgical Approach to Induce Endolymphatic Hydrops in the Guinea Pig

几内亚猪诱导内部水解的修正手术方法

Endolymphatic hydrops is an enlargement of scala media that is most often associated with Meniere&#39;s disease, though the pathophysiologic mechanism(s) ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

神经干细胞向大鼠和小鼠大脑的动脉内传递：对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

立体定位手术方法 ，通过 大水池在小鼠中显微注射尾部脑干和上颈椎脊髓

立体定位手术方法，通过大水池在小鼠中显微注射尾部脑干和上颈椎脊髓