成像神经胶质瘤启动<em在体内</em>通过抛光和增强的薄膜头骨的颅窗
Summary
通过结合抛光和增强的薄的头骨（端口）的颅窗和胶质母细胞瘤（GBM）细胞注射，我们可以观察到活老鼠大脑中的纵向注入GBM细胞胶质瘤的萌生和扩展。
Abstract
神经胶质瘤是最致命形式的人类癌症之一。最有效的神经胶质瘤治疗，手术日期，然后通过辐射处理，为患者提供只有温和的好处，因为大多数患者没有生存超过5年诊断后，由于神经胶质瘤复发1,2。在人脑肿瘤中的癌干细胞的发现带来了希望有新的治疗策略的发展产生巨大的影响脑胶质瘤3。癌症干细胞是指通过他们的自我更新和分化能力，被认为是唯一的肿瘤细胞有能力启动新的肿瘤4。神经胶质瘤复发放射治疗后产生的电阻（GSCS）治疗脑胶质瘤干细胞被认为是5-10。 在体内 ，的GSCS是居住在血管周围是很重要的利基，为保持其干细胞样特性11-14 。中央的organization的GSC利基的血管内皮细胞12。现有证据表明GSCS及其与血管内皮细胞的肿瘤的发展是重要的，和拣选GSCS及其与内皮细胞作为重要的治疗目标胶质瘤。实验确定他们的能力，引发新的肿瘤，在原位肝移植15的存在GSCS。这通常是通过注入GBM细胞的特定数目的从人类肿瘤中分离到的严重的免疫缺陷小鼠的大脑，或鼠标GBM细胞到同源宿主小鼠的大脑。检测肿瘤的生长足够的时间，让GSCS注入GBM细胞之间引起新的肿瘤，通常几个星期或几个月后进行。因此，现有的检测不允许从单一的GSCS 体内肿瘤的发生过程中的重要病理检查。因此，基本我nsights到GSCS和它们的相互作用，与血管内皮细胞的肿瘤发生的早期阶段的具体作用缺乏。这种见解是开发新的治疗脑胶质瘤的策略至关重要，并且将有很大的影响，对防止脑胶质瘤复发的患者。在这里，我们已经适应了端口颅窗口程序16和体内双光子显微镜，让可视化注入的的GBM细胞在活老鼠的大脑肿瘤的发生。我们的技术铺平了道路，为未来努力澄清的关键信号传导机制之间的GSCS和血管内皮细胞在神经胶质瘤开始。
Protocol
Discussion
一个成功的端口颅窗的关键是减薄和抛光。虽然最初的细化，可以迅速执行，护理应采取措施，确保在大面积同质的头骨变薄。我们通常涂上一层薄薄的生理盐水到颅骨，然后薄颅骨一通的时间，使得生理盐水溶液蒸发后不久的微钻头已经越过颅骨一次。这让我们缓慢而均匀薄头骨。在显微镜下是一个稳定的手也很关键。一旦，颅骨变薄接近预期的厚度，我们通常使用金刚石研磨膏打磨变薄颅骨1…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作是由杰克逊实验室癌症中心的试点格兰特和美国缅因州癌症基金会的支持。
Materials
| Name of reagent | Company | Catalogue number | Comments (optional) |
| DMEM/F12(1:1) with Sodium Pyruvate | Thermo Sci Hyclone | SH3026101 | |
| B-27 Serum Free Supplement (50x) | Invitrogen | 17504-044 | |
| GlutaMax-1 Supplement | Invitrogen | 35050061 | |
| Penicillin-Streptomycin Solution | Thermo Sci Hyclone | SV30010 | |
| Accutase-Enzyme Cell Detachment Medium | eBiosciences | 00-4555-56 | |
| T25 flasks-vent cap green | SARSTEDT | 83.1810.502 | |
| 70% alcohol | JAX LAHS | ||
| 10% povidone-iodine topical solution | JAX LAHS | ||
| Ketamine HCl | Butler Animal Health Supply | NDC# 11695-0550-1 | |
| Xylazine | Akorn, Inc. | NADA# 139-236 | |
| Carprofen | JAX LAHS | ||
| Ophthalmic ointment | Dechra Veterinary Products | 17033-211-38 | |
| 0.5% Lidocaine HCl | REGENT, Inc. | NDC 0517-0625-25 | |
| Cyanoacrylate glue | Henkel Corp. | 46551 | |
| Sterile Swabs | Fisher Scientific | 23-400-114 | |
| Diamond paste | Widget Supply | BBE60 | |
| Tin oxide | LORTONE, Inc. | 591-038 | |
| Liner Bond 2V | KURARAY Medical Inc. | 1921-KA | |
| Clearfil AP-X | KURARAY Medical Inc. | 1721-KA | |
| Saline | JAX LAHS | ||
| Cover glass | Warner Instruments | 64-0720 | |
| Hex Nut | Small Parts, Inc. | HNX-0090-C | |
| Syringe Pump | Syringepump.com | NE-1000 | |
| Two-Photon imaging system | Custom built (Any commercial system would work) |
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