外切囊梗死模型使用光化学技术
Summary
这份手稿描述囊梗塞的建模技术。在这里，我们利用与手术后前目标映射光的低强度改良光化学技术。使用这种技术，我们创造了持续性运动障碍外接囊梗死模型。
Abstract
Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.
Introduction
直到最近，“灰质行程（GMS）模型”已经被专门用来了解中风的病理生理机制，并指导新治疗方法的发展。然而，出现了行程的患病率增加，影响在老年个体，这构成15皮层下白质-所有笔划1,2的25％。大量的研究已经进行使用GMS模型对中风，而有一些已经使用白质行程（WMS）模型的研究很少。在啮齿类动物中白质比在人类或灵长类白质基本上更少。因此，更难以在脑白质3选择性地访问和破坏靶区域。此外，没有有效的工具已经被开发迄今为止为选择性地破坏的靶向白质的计划的程度。因此，一直存在缺乏脑白质笔划研究适当的模型。
动物ST洛克模型经常用于监测马达恢复的新康复和治疗方法的发展的进展。它是理想的，利用表现出一个长期的神经功能缺损一致与人类中风4,5展示了解剖改变的动物模型。在这方面，马达赤字和脑以下梗塞损毁的广泛参与快速恢复可能不追求中风研究的现实的。以前荚膜梗塞模型已被制成由内皮素-1（ET-1）的内部颈动脉或脉络膜前动脉和扩散的闭塞进入内囊6-9。尽管如此，动脉闭塞，需要动脉小心剥离，但它产生的梗塞病灶的面积广，包括内囊，而不持久行为缺陷。此外，ET-1是不是扩散彻底摧毁内囊后肢，因此不太明显或持续BEHavioral赤字。
一个光化学梗死模型已被广泛地用于产生各种类型的皮质梗塞病灶和皮层下结构10的。该技术包括静脉内给药后焦距照明，这导致在小血管的血小板凝集和生成梗塞病灶10。光化学技术已被广泛使用，以创建GMS病变，而它很少被用来产生WMS病变5,11。对于这种技术，玫瑰红染料和光照射的组合已被证明是在靶结构的破坏是有用的，从而导致相应的功能缺损。的光化学技术的关键因素是光照射，因为它决定了梗塞病灶的大小。光照射结果在灰质和白质不同的效果，由于光线的散射是白龙马高出4倍tter灰质比12;因此，如果光强度具有足够低辐照度（<1,140毫瓦/毫米2），一个能限制扩展到光化学病变影响的程度的白质（ 即 ，内囊）。例如，高能量的光可以诱导两个灰质和白质梗死，但是较低的能量光只能在白质诱导photothrombosis。另外，光能量的穿透是非常有限的。光能量大约99％失去了从外光13源1毫米。因此，预期能够准确定位，低能量的光只在与邻近的灰质的最小侵入白质诱导photothrombosis。
在这里，我们描述了一种新方法，在啮齿类动物中的内囊的前肢区域来创建梗塞病灶。我们描述的前肢区域的识别方法在内部CApsule，光照射，包括调整与光的传送，和梗塞病灶的生成的技术。我们还描述了用于评估囊建模的完整性行为测试。
Protocol
Representative Results
Discussion
这里提出的囊梗死模型表明，在前肢的功能显着和持久的运动障碍有针对性的病变。皮质下囊中风之前的机型都表现出了足够的程度运动功能障碍和快速恢复过程6,8,9的。在这个意义上，该模型类似于表现出长期的功能障碍的临床荚膜梗塞例。
在一个外接荚膜梗塞模型的发展中的最重要的步骤是：1），以正确地识别旨在禁止内部胶囊内的功能的主体部的躯体表示; 2）…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作是由来自医疗系统工程研究所（IMSE）从GIST GIST加州理工学院联合基金（K04592）的资助，并通过韩国NRF的基础科学研究计划由科学，信息和通信技术及未来规划部提供资金支持（NRF-2013R1A2A2A01067890）。
Materials
| DC Temperature controller | WORLD PRECISION INSTRUMENTS, INC. | ATC1000 | |
| Digital Stereotaxic Instruments | STOELTING CO. | 51900 | |
| Electrical Stimulator | CyberMedic Corp. | EMGFES 2000 | |
| Epoxy | Precision Fiber Products, INC. | PFP-353ND1 | Mix Ratio: 10(A):1(B-hardener) by weight Curing Schedule: 1 minute @150°C 2~5 minutes @120°C 5~10 minutes @100°C 15~30 minutes @80°C |
| Fiber Optic Scribe | THORLABS, INC | S90R | |
| Fiber patch cable | KOREA OPTRON Corp. | Outer diameter: 3mm Ø200 µm 0.39 NA FC/PC-FC/PC 1 m |
|
| Laser Power Supply | CHANGCHUN NEW INDUSTRIES OPTOELECTRONICS TECH. CO., LTD. | MGL-FN-532nm-200mW-14010196 | |
| Crimp ring | DAWOOTECH CO.,LTD. | Length: 19mm Inner diameter: 3mm Outer diameter: 3.8mm Material: SUS |
|
| Micro4-micro syringe pump controller | WORLD PRECISION INSTRUMENTS, INC | 95100 | |
| Optical Power Meter | THOLABS, INC | PM100D | |
| Paraformaldehyde | SIGMA-ALDRICH CO. LLC. | P6148 | |
| Diamond lapping (polishing) sheet | THORLABS, INC | LF3D | Grit : 3 µm |
| Diamond lapping (polishing) sheet | THORLABS, INC | LF6D | Grit : 6 µm |
| Rose Bengal | SIGMA-ALDRICH CO. LLC. | 330000 | |
| Needle for spinal anesthesia with pencil point tip (Spinal needle) | B.BRAUN MELSUNGEN AG | 4502027 | Size: 27G Length: 88mm Needle: 0.40mm |
| Waterproof sandpaper | DEERFOS CO.,LTD | CC261 | Grit : 1000 µm |
| Nanofil 10uL syringe | WORLD PRECISION INSTRUMENTS, INC | NANOFIL | |
| Nanofil 33G BVLD needle | WORLD PRECISION INSTRUMENTS, INC | NF33BV-2 | |
| AAV-GFP virus | UNC Vector Core | AAV2-CamKIIa-eYFP | 2×10^12 virus molecules/ml |
| Anti-Green Fluorescent Protein, Rabbit IgG fraction | Life Technologies, INC | A11122 | primary antibody (1:200) |
| Goat Anti-Rabbit IgG (H+L) | Life Technologies, INC | A11034 | secondary antibody (1:500) |
| Ceftezole | GUJU Pharma CO.,LTD. | A27802741 | 0.1%, 1ml |
| Lidocain hydrochloride injection | JEIL PHARMACEUTICAL CO.,LTD. | A04900271 | 2%, 1ml |
| Hand Piece Drill | Seshin | ||
| Digital optical power and energy meter | THORLABS, INC | PM100D | |
| Ketopropen | UNIBIOTech |
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