异位黏膜移植程序直接给药到小鼠的大脑
Summary
已开发人力内镜颅底重建的小鼠模型，会使用鼻腔粘膜移植大脑和鼻子之间的半透界面。这种方法使研究人员能够研究发货到全身给药时，被排除在外，否则由血脑屏障高分子量治疗中枢神经系统。
Abstract
递送治疗剂进入脑是通过血 – 脑屏障（BBB）的制约的极性和高分子量化合物从血液进入脑组织的通道的存在阻碍。一些直接交付的成功在人类已通过经颅导管植入已经实现;然而，这种方法具有很强的侵袭，并与众多的并发症。侵入性较低的替代办法是通过外科手术植入，半透膜如用于下列在人类鼻内镜切除肿瘤手术，以修复颅底缺损鼻黏膜剂量的大脑。药物传递虽然这种膜能有效地绕过血脑屏障，并直接扩散到脑和脑脊液。通过这种方法的启发，在小鼠的手术方法的开发，使用捐赠者间隔粘膜栽种了一个多颅外手术血脑屏障的缺陷。该模型已被证明能有效地允许的高分子量化合物的通路进入大脑。由于众多的候选药物都无法穿越血脑屏障，这种模式是对神经系统和精神疾病进行的新疗法临床前试验的价值。
Introduction
神经和精神疾病的治疗中是受到严重的血-脑屏障（BBB），防止所有潜在的药物制剂的95％以上到达中枢神经系统1-3的存在阻碍。例如，神经胶质源性神经营养因子（GDNF）已被证明可有效地治疗帕金森氏病时，直接注射到大脑，交付时全身性，因为它不能穿透血脑屏障4-6不过是无效的。
众多走近已经发展到试图绕过这个问题。改善全身递送neurotheraputics的已证实通过使用含有抗体的选择性为位于脑毛细血管内皮转运蛋白药物结合物;然而这种方法并没有被证明是适用于范围广泛的药物7,8。此外，血脑屏障渗透性开放已经临床使用盟友，但这种方法从全身用药剂量患有而不是一个更直接的传递到感兴趣的9大脑区域。大量的努力已投入在优化直接针对大脑10-12的希望鼻交付。虽然有些已经取得成功的，结论性的结果只获得针对药物具有内源性受体，如胰岛素13,14。此外鼻交付机制一直存在争议证据通过嗅觉神经元摄取或通过血液11暗示间接进入大脑。使用植入导管直接的，经颅传递已经实现，但这一程序具有很强的侵袭，并与众多的并发症15,16关联。迄今为止，不存在一般的微创方法，以提供高分子量的化合物进入脑。
本文中所呈现的是一种鼠的外科手术创建与大脑半透界面。这是通过移植粘膜外植体17以上的小鼠外科开颅缺陷来实现的。使用此程序它已经表明，可溶性化合物高达500 kDa的可递送到中枢神经系统（直接进入脑实质以及进入脑脊液）在两个时间和分子量依赖性方式18。绕过血脑屏障这种方法是一款型号为颅底缺损修复的人类，它使用血管黏膜瓣下鼻内镜手术19,20修复颅骨孔。
Protocol
Representative Results
Discussion
本文所描述的过程中最困难的步骤是一个足够大的粘膜的成功转移到脑表面。这一步是由显著容易，如果收获的鼻中隔足够大，打扫好。如果隔膜的腹侧部分被截断，应获得一个新的移植物。钻孔角度应垂直于鼠标头，以确保粘膜不被钻头损坏。如果宽度大于推荐钻孔通道被接便更难除去，其连接到该隔膜的额外的组织。任何大块的结缔组织应该用锋利的剪刀割伤，而不是试图拉他们松被删除。?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是由该Mcihael J.福克斯基金会帕金森研究2011年快速响应创新奖励计划。资助者在研究设计，数据收集和分析，发布的决定，或准备稿子没有任何作用。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| mice | Taconic | C57BL/6 | |
| Isoflurane | Piramal Healthcare | ||
| Student Fine Scissors | Fine Science Tools | 91461-11 | |
| pneumatic drill | MTI Dental | 333-CB | |
| drill bit | |||
| forcepts | Fine Science Tools | 91106-12 | |
| 0.9% Sodium Chloride Injection USP | Abbott Laboratories | 4925 | |
| Polystyrene Detri Dish | Fisher | 08-757-12 | for temporarily storing graft |
| Bead Sterilizer | Fine Science Tools | 18000-45 | |
| Oxygen/Isoflurane System | SurgiVet | V720100 | |
| Temperature Control System | Physitemp | TCAT-2LV | |
| Small Animal Stereotaxic Instrument | KOPF | Model 940 | |
| Eye Ointment | |||
| Electric shaver | |||
| Cotton-Tipped Applicators | Fisher | 23-400-106 | |
| 7.5% Provadone Iodine | Betadine surgical scrub | ||
| 70% Ethanol | |||
| Surgical Blade Stainless | Feather | 2976#10 | |
| Scalpel Handle – #3 | Fine Science Tools | 10003-12 | |
| 3% Hydogen Peroxide | for cleaning the skull | ||
| Vetbond Tissue Adhesive | 3M | 1469SB | |
| needles | Becton, Dickinson and Company | 305176 | needle tip cut off and used as well |
| syringes | Becton, Dickinson and Company | 309597 | |
| nitrile gloves | Denville Scientific Inc | G4162 | for well closure and protection of graft |
| 5-0 Nylon Suture Thread | |||
| Student Halsey Needle Holder | Fine Science Tools | 91201-13 | |
| cyanoacrylate adhesive | commecially available super glue | ||
| Dental Cement Kit, 1 lb, Pink Opaque | Stoelting | 51458 | |
| isobutane (2-methylbutane) | Aldrich | M32631 | for dry ice bath |
| dry ice |
References
- Cardoso, F. L., et al. Looking at the blood-brain barrier: Molecular anatomy and possible investigation approaches. Brain Res. Rev. 64, 328-363 (2010).
- Pardridge, W. M. Drug transport across the blood-brain barrier. J. Cereb. Blood Flow Metab. 32, 1959-1972 (2012).
- Chen, Y., Liu, L. Modern methods for delivery of drugs across the blood-brain barrier. Adv. Drug Deliv. Rev. 64, 640-665 (2012).
- Cheng, F. -. C., et al. Glial cell line-derived neurotrophic factor protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in C57BL/6 mice. Neurosci. Lett. 252, 87-90 (1998).
- Grondin, R., et al. controlled GDNF infusion promotes structural and functional recovery in advanced parkinsonian monkeys. Brain. 125, 2191-2201 (2002).
- Kirik, D., et al. Localized striatal delivery of GDNF as a treatment for Parkinson disease. Nat. Neurosci. 7, 105-110 (2004).
- Pardridge, W. M. Drug and gene targeting to the brain with molecular trojan horses. Nat. Rev. Drug Discov. 1, 131-139 (2002).
- Pardridge, W. M. Blood-brain barrier delivery of protein and non-viral gene therapeutics with molecular Trojan horses. J. Control. Release. 122, 345-348 (2007).
- Bellavance, M. -. A., et al. Recent advances in blood-brain barrier disruption as a CNS delivery strategy. AAPS J. 10, 166-177 (2008).
- Merkus, F. H. M., Berg, M. Can nasal drug delivery bypass the blood-brain barrier. Drugs R. D. 8, 133-144 (2007).
- Dhuria, S. V., et al. Intranasal delivery to the central nervous system: Mechanisms and experimental considerations. J. Pharm. Sci. 99, 1654-1673 (2010).
- Illum, L. Nasal drug delivery-possibilities, problems and solutions. J. Control. Release. 87, 187-198 (2003).
- Craft, S., et al. Intranasal insulin therapy for alzheimer disease and amnestic mild cognitive impairment: A pilot clinical trial. Arch. Neurol. 69, 29-38 (2012).
- Freiherr, J., et al. Intranasal insulin as a treatment for Alzheimer’s Disease: A review of basic research and clinical evidence. CNS Drugs. 27, 505-514 (2013).
- Gill, S. S., et al. Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease. Nat. Med. 9, 589-595 (2003).
- Love, S., et al. Glial cell line-derived neurotrophic factor induces neuronal sprouting in human brain. Nat. Med. 11, 703-704 (2005).
- Antunes, M. B., et al. Murine nasal septa for respiratory epithelial air-liquid interface cultures. BioTechniques. 43, 195-204 (2007).
- Bleier, B. S., et al. Permeabilization of the blood-brain barrier via mucosal engrafting: implications for drug delivery to the brain. PLoS ONE. 8, (2013).
- Bernal-Sprekelsen, M., et al. Closure of cerebrospinal fluid leaks prevents ascending bacterial meningitis. Rhinology. 43, 277-281 (2005).
- Bleier, B. S., et al. Laser-assisted cerebrospinal fluid leak repair: An animal model to test feasibility. Otolaryngol. Head Neck Surg. 137, 810-814 (2007).
