精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像
Summary
该协议描述了一种慢性颅窗植入技术，可用于神经-胶质-血管结构、相互作用和功能在健康和患病条件下的纵向成像。它是颅内成像方法的补充替代品，虽然通常倾向于，但具有一些关键限制。
Abstract
中枢神经系统（CNS）由神经元、胶质细胞、频闪细胞和血管细胞的复杂相互作用调节，促进其正常功能。虽然在体外或体外一起单独研究这些细胞提供了有用的生理信息;在这样的语境中，神经细胞生理学的显著特征将会被遗漏。因此，有必要研究神经细胞在原生体内环境。此处详述的协议描述了啮齿动物皮层中神经细胞的重复体内双光子成像，作为在从数小时到数月的较长时间段内可视化和研究特定细胞的工具。我们详细描述使用严重稳定的脑血管作为粗糙的地图或荧光标记的树突作为特定大脑区域感兴趣的精细地图。使用这些映射作为视觉键，我们展示神经细胞如何精确重新定位，以便随后进行重复的体内成像。使用荧光标记微胶质、神经元和NG2+ 细胞的体内成像示例，该协议演示了该技术在较长时间段内允许在同一大脑位置重复可视化细胞动力学的能力，这进一步有助于理解这些细胞在正常生理学或病理侮辱中的结构和功能反应。必要时，此方法可与神经细胞的功能成像相结合，例如，与钙成像相结合。当基因小鼠模型或具有不同荧光标签的特定染料可用于标记感兴趣的细胞时，这种方法尤其具有一种强大的技术，用于可视化体内不同细胞类型中 CNS 的物理相互作用。
Introduction
中枢神经系统（CNS）受各种居民细胞类型（包括神经元、胶质细胞和血管相关细胞）之间相互作用的复杂相互作用的支配。传统上，神经细胞研究在分离，共培养1,21，2，3，4，5（体外）或切除脑组织（体外,3,4,5）6，7，8，9，10个上下文。6,7,8,9,10然而，需要进一步了解神经细胞行为和相互作用在原生环境中的完整的大脑体内。在此协议中，我们描述了一种在体内绘制感兴趣区域并在未来成像会话中精确重新成像这些区域的方法，以跟踪各种 CNS 细胞类型之间长时间的复杂相互作用。
体内成像方法的发展为正确理解神经,功能,11、12、13、14、15,1213,14提供了显著收益。具体来说，与传统的体外和体外方法相比，这些方法具有若干优势。首先，体内成像系统具有生理相关的细胞和组织成分，如血管与细胞相互作用的完整剧目，以提供神经网络生理学的全面了解。其次，最近的发现表明，当从原生环境中去除时，某些神经细胞（如微胶质）会失去其特性的重要特征，因此生理特性16，17,17可以保留在体内环境中。第三，体内成像系统为数周至数月的稳定纵向调查提供了研究CNS细胞相互作用的机会。最后，鉴于越来越多的证据表明来自周围免疫系统18、19和19中枢神经系统生理学中的微生物群20、21,21的贡献，体内系统提供了一个平台来询问这些贡献和对CNS细胞的影响。因此，使用纵向体内成像来研究健康、受伤和患病状态的神经免疫生理学和相互作用的方法是传统方法的一大补充。
在这个协议中，我们描述了一种可靠的方法，以图像大脑中不同的细胞类型，包括微胶质，神经元和NG2+细胞为例。开发了两种在体内可视化神经细胞的方法：瘦头骨方法和颅窗法的开放头骨。虽然瘦身的头骨方法正在使用，是首选，因为他们克服了开放性头骨方法的一些缺点，如胶质细胞活化，高于生理的脊椎动力学和使用抗炎剂22，23，24，25，瘦化头骨方法也显示了一些关键缺点。22,23,24,25首先，变薄过程是一个非常微妙的程序，许多研究人员发现很难完善，尤其是当重新变薄是必要的。这种情况之所以如此，是因为实验者往往很难确定他们把头骨稀释到±20μm的深度。其次，为了在小鼠之间进行适当的比较，变薄需要是相同的，成像会话或小鼠之间的各种变薄成功可能会使神经结构的可视化复杂化。第三，当用于纵向成像时，头骨变薄的动物只能在头骨重新变薄时用于数量有限的会话。第四，由于一些骨组织仍然存在，成像深度的清晰度可能会从薄的头骨方法中受损，从而能够对更肤浅但更深的区域进行出色的可视化。有鉴于此，更深的大脑结构，如海马，不能成功地用薄头骨的方法成像。这些考虑提出了需要采取替代和补充办法，以克服这些关切。
除了薄头骨方法外，开放的头骨窗口植入方法使用用光学透明玻璃盖玻片替换头骨的程序。这允许几乎无限数量的成像会话。此外，鉴于用玻璃盖玻片替换头骨，这种方法允许在数小时到数月的长时间内对荧光标记脑细胞进行清晰的观察，因此，可用于研究与生理学、衰老和病理学相关的细胞活动和相互作用。
总的来说，我们详细介绍了通过立体轴颅切除术植入慢性颅骨窗口的步骤，这种颅骨切除术使感兴趣的大脑区域在体内成像。我们还描述了如何利用严重稳定的脑血管或荧光标记的树突分别生成感兴趣的大脑区域的粗糙或精细图。然后，此方法可用于多个会话的重复映像。因此，这种技术的重要性在于它能够成像大脑元素的长期变化或停滞，包括不同细胞类型的排列、形态和相互作用。
Protocol
Representative Results
Discussion
体内双光子成像的出现为探索健康大脑中过多的细胞相互作用和动态提供了机会。初步研究的重点是使用开放颅骨切除术的方法，通过急性和慢性成像37，38图像神经元树突图像。这也可以用来阐明大脑中的神经免疫相互作用。该协议描述了一种短期或长期对荧光标记细胞（特别是微胶质，大脑的常驻免疫细胞）进行可靠成像的方法。使用染料标记的血?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 Eyo 实验室的成员讨论本手稿中提出的想法。我们感谢弗吉尼亚大学基普尼斯实验室的贾斯汀·鲁斯滕霍芬博士为NG2DSRed小鼠33的礼物。这项工作得到了国家卫生研究所神经疾病和中风研究所的资助，为U.B.E（K22 NS104392）。
Materials
| Coverglass (3mm) | Warner Instruments | 64-0726 | |
| Cyanoacrylate glue (Krazy Glue) | Amazon | https://www.amazon.com/Krazy-Glue-Original-Purpose-Instant/dp/B07GSF31WZ/ref=sr_1_2?keywords=krazy+glue&qid=1583856837&s=pet-supplies&sr=8-2 | |
| Demi Ultra LED Curing Light System | Dental Health Products, Inc | 910860-1 | |
| Dental Drill | Osada: www.osadausa.edu | EXL-M40 | |
| Drill Bit | Fine Science Tools | #19008-07 | |
| Eye Ointment | Henry Schien | 1338333 | |
| iBond Total Etch (Primer glue) | Chase Dental Supply (Heraeus Kulzer) | 66040094 | |
| Rhodamine B | Millipore Sigma | 81-88-9 (R6626) | |
| Tetris Evoflow glue (Final glue) | Top Dent (Ivoclar Vivadent) | #595956 | |
| Wahl Brav Mini+ | Amazon | https://www.amazon.com/Wahl-Professional-Animal-BravMini-41590-0438/dp/B00IN24ILE/ref=asc_df_B00IN24ILE/?tag=hyprod-20&linkCode=df0&hvadid=167141013968&hvpos=&hvnetw=g&hvrand=12368793083893626704&hvpone=&hvptwo=&hvqmt=&hvdev=c&hvdvcmdl=&hvlocint=&hvlocphy=9008337&hvtargid=pla-332197544154&psc=1 |
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