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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 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?…
Representative Results
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<…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了R01 NS079623，P01 HL149630和P01 HL095491的支持。
Materials
| Alcohol pad | Med-Vet International | SKU: MDS090735Z | skin preparation for the prevention of surgical site infection |
| Angled forceps, Dumont #5/45 | FST | 11251-35 | only to grab dura |
| Betadine pad | Med-Vet International | SKU:PVP-PAD | skin preparation for the prevention of surgical site infection |
| Cholera toxin subunit-b, Alexa Fluor 488/594 conjugate | Thermo Fisher Scientific | 488: C34775, 594: C22842 | Fluorescent tracer |
| Clippers | Wahl | Model MC3, 28915-10 | for shaving fur at surgical site |
| Electrode holder with corner clamp | Kopf | 1770 | to hold glass pipette |
| Flowmeter | Gilmont instruments | model # 65 MM | to regulate flow of isoflurane and oxygen to mouse on the surgical plane |
| Fluorescent microspheres, polystyrene | Thermo Fisher Scientific | F13080 | Fluorescent tracer |
| Heating pad | Stoelting | 53800M | thermoregulation |
| Induction chamber with port hook up kit | Midmark Inc | 93805107 92800131 | chamber providing initial anasthesia |
| Insulin Syringe | Exelint International | 26028 | to administer saline and analgesic |
| Isoflurane | Med-Vet International | SKU:RXISO-250 | inhalant anesthetic |
| Isoflurane Matrix VIP 3000 vaporizer | Midmark Inc | 91305430 | apparatus for inhalant anesthetic delivery |
| Laminectomy forceps, Dumont #2 | FST | 11223-20 | only to clean dura |
| Medical air, compressed | Linde | UN 1002 | used with stimulator & PicoPump for providing air for precision solution injection |
| Meloxicam SR | Zoo Pharm LLC | Lot # MSR2-211201 | analgesic |
| Microhematocrit borosilicate glass pre calibrated capillary tube | Globe Scientific Inc | 51628 | for transfection of material to designated co-ordinates |
| Mouse adaptor | Stoelting | 0051625 | adapting rat stereotaxic frame for mouse surgery |
| Needle holder, Student Halsted- Mosquito Hemostats | FST | 91308-12 | for suturing |
| Oxygen regulator | Life Support Products | S/N 909328, lot 092109 | regulate oxygen levels from oxygen tank |
| Oxygen tank, compressed | Linde | USP UN 1072 | provided along with isoflurane anasthesia |
| Plastic card | not applicable | not applicable | any firm plastic card, cut to fit the stereotactic frame (e.g. ID card) |
| Pneumatic PicoPump ( or similar) | World Precision Instruments (WPI) | SYS-PV820 | For precision solution injection |
| Saline, sterile | Mountainside Medical Equipment | H04888-10 | to replace body fluids lost during surgery |
| Scalpel handle, #3 | FST | 10003-12 | to hold scalpel |
| Scissors, Wagner | FST | 14070-12 | to cut polypropylene suture |
| Spring scissors, Vannas 2.5mm with accompanying box | FST | 15002-08 | scissors only to open dura, box to elevate body |
| Stereotactic micromanipulator | Kopf | 1760-61 | attached to electrode holder to adjust position based on co-ordinates |
| Stereotactic 'U' frame assembly and intracellular base plate | Kopf | 1730-B, 1711 | frame for surgery |
| Sterile cotton tipped applicators | Puritan | 25-806 10WC | absorbing blood from surgical field |
| Sterile non-fenestrated drapes | Henry Schein | 9004686 | for sterile surgical field |
| Sterile opthalmic ointment | Puralube | P1490 | ocular lubricant |
| Stimulator & Tubing | Grass Medical Instruments | S44 | to provide controlled presurred air for precision solution injection |
| Surgical Blade #10 | Med-Vet International | SKU: 10SS | for skin incision |
| Surgical forceps, Extra fine Graefe | FST | 11153-10 | to hold skin |
| Surgical gloves | Med-Vet International | MSG2280Z | for asceptic surgery |
| Surgical microscope | Leica | Model M320/ F12 | for 5X-40X magnification of surgical site |
| Suture 5-0 polypropylene | Oasis | MV-8661 | to close the skin |
| Tegaderm | 3M | 3M ID 70200749250 | provides sterile barrier |
| Universal Clamp and stand post | Kopf | 1725 | attached to stereotactic U frame and intracellular base plate |
| Wound hook with hartman hemostats | FST | 18200-09, 13003-10 | to separate muscles and provide surgical window |
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Cite This Article
Joshi, K., Kirby, A., Niu, J., VanderHorst, V. Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice. J. Vis. Exp. (179), e63344, doi:10.3791/63344 (2022).