直接插管植入猪的蓄水池
Summary
本文介绍了直接将套管植入猪大水池的分步方案。
Abstract
淋巴系统是大脑中的废物清除系统，依赖于星形胶质细胞结合的血管周围空间中脑脊液（CSF）的流动，并且与神经毒性肽（如β淀粉样蛋白）的清除有关。淋巴功能受损加剧了神经退行性疾病（如阿尔茨海默氏症）动物模型中的疾病病理学，这突出了理解这种清除系统的重要性。淋巴系统通常通过大水池插管（CMc）进行研究，其中示踪剂直接输送到脑脊液（CSF）中。然而，大多数研究都是在啮齿动物身上进行的。在这里，我们展示了CMc技术在猪中的适应性。在猪中使用CMc，可以在回脑大脑中以高光学分辨率研究淋巴系统，从而弥合啮齿动物和人类淋巴动物之间的知识差距。
Introduction
脑脊液（CSF）是一种血液超滤液，存在于中枢神经系统（CNS）内和周围1，2。除了为大脑提供浮力或吸收破坏性的机械力外，脑脊液在清除CNS3代谢废物方面也起着关键作用。最近表征的淋巴系统促进了废物清除，该系统允许脑脊液通过血管周围空间（PVS）通过脑实质的对流流，该血管周围空间包围穿透性动脉3，4，5。该过程已被证明依赖于水通道蛋白-4（AQP4），AQP4是主要在星形细胞末端上表达的水通道，与PVS4，6结合。在将荧光/放射性示踪剂或造影剂引入CSF7，8，9，10，11之后，使用先进的光学显微镜或磁共振成像（MRI）通过体内和离体成像实现淋巴系统的研究。
在不损害脑实质的情况下将示踪剂引入脑脊液的有效方法是通过大水池插管（CMc）12，13。到目前为止，绝大多数淋巴研究都是在啮齿动物中进行的，并且在高等哺乳动物中避免了，因为CMc的侵入性加上与小型哺乳动物一起工作的实际简单性。此外，小鼠的薄头骨允许在不需要颅窗的情况下进行 体内 成像，随后允许简单的脑提取11，14。在人类中进行的实验已经产生了关于淋巴功能的有价值的宏观 体内 数据，但依赖于腰椎远端的鞘内示踪剂注射，此外，利用MRI不能产生足够的分辨率来捕获淋巴系统的显微解剖法7，15，16.了解高等哺乳动物中淋巴系统的结构和范围对于将其转化为人类至关重要。为了促进淋巴细胞向人类的转化，重要的是将啮齿动物中进行的技术应用于高等哺乳动物，以便能够直接比较不同认知和大脑复杂性增加的物种的淋巴系统17。猪和人的大脑是回脑的，具有折叠的神经结构，而啮齿动物的大脑是脑畸形的，因此彼此之间有很大的差异。就整体大小而言，猪的大脑也更可与人类相媲美，比人类大脑小10-15倍，而小鼠大脑小3000倍18。通过更好地了解大型哺乳动物的淋巴系统，有可能利用人类淋巴系统进行中风，创伤性脑损伤和神经变性等疾病的未来治疗干预。体内猪中的直接CMc是一种允许 在 高等哺乳动物中对淋巴系统进行高分辨率光学显微镜检查的方法。此外，由于所用猪的大小，可以应用类似于人类手术中使用的监测系统，从而可以严格记录和调节重要功能，以评估这些功能如何促进淋巴功能。
Protocol
Representative Results
Discussion
本文描述了在猪中执行大蓄水池直接插管的详细方案，包括必要的制备、外科手术、示踪剂输注和脑的提取。这需要具有与大型动物打交道的经验和认证的人。如果正确执行，这允许将具有保证的所需分子直接递送到CSF中，之后可以使用一系列不同的高级光成像模式来探索大型哺乳动物中高分辨率的CSF分布和淋巴功能。
重要的是要注意，尽管这与啮齿动物的蓄水池大插管相?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了Knut和Alice Wallenberg基金会，Hjärnfonden，Wenner Gren基金会和Crafoord基金会的支持。
Materials
| 0.01% azide in PBS | Sigmaaldrich | S2002 | |
| 18G needle | Mediq | ||
| 1ml Syringe | FischerSci | 15849152 | |
| 20G cannula | Mediq | NA | |
| 22G cannula | Mediq | NA | |
| 4% paraformaldehyde | Sigmaaldrich | P6148 | |
| Anatomical forceps | NA | NA | |
| Bovine serum albumin Alexa-Fluor 647 Conjugate | ThermoFischer | A34785 | 2 vials (10mg) |
| CaCl2 | Sigmaaldrich | C1016 | |
| Chisel | ClasOhlson | 40-8870 | |
| Dental cement | Agnthos | 7508 | |
| compact saw | ClasOhlson | 40-9517 | |
| Glucose | Sigmaaldrich | G8270 | |
| Hammer | ClasOhlson | 40-7694 | |
| Insta-Set CA Accelerator | BSI-Inc | BSI-151 | |
| IV line TAP, 3-WAYS with 10cm extension | Bbraun | NA | |
| KCl | Sigmaaldrich | P9333 | |
| Marker pen | NA | NA | |
| MgCl2 | Sigmaaldrich | M8266 | |
| MilliQ water | NA | NA | |
| NaCL | Sigmaaldrich | S7653 | |
| NaH2PO4 | Sigmaaldrich | S8282 | |
| NaHCO3 | Sigmaaldrich | S5761 | |
| No. 20 scalpel blade | Agnthos | BB520 | |
| No. 21 Scalpel blade | Agnthos | BB521 | |
| No. 4 Scalpel handle | Agnthos | 10004-13 | |
| Saline | Mediq | NA | |
| Salmon knife | Fiskers | NA | |
| Self-retaining retractors | NA | NA | |
| Superglue | NA | NA | |
| Surgical curved scissors | NA | NA | |
| Surgical forceps | NA | NA | |
| Surgical towel clamps | NA | NA |
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