玫瑰红Photothrombosis共聚焦光学成像<em>在体内</em>：单支脑卒中模型
Summary
Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.
Abstract
In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel.
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.
Introduction
体内细胞应答的描述的技术允许可视化之后立即感应玫瑰红的photothrombosis在一个完整的小鼠。玫瑰红（4,5,6,7-四氯-2'，4'，5'，7'-四碘）是用于诱导在动物模型缺血性中风（小鼠和大鼠）的光敏染料。通过减薄头骨与564纳米的激光以下推注的RB的尾静脉和随后的照明，血栓诱导引起生理行程1。该方法最初是由布鲁姆和El-萨班在1977年描述，并通过华生在20世纪80年代中期以后1,2改编。简言之，玫瑰红照射绿色激发光（在我们的情况下，561纳米的激光），其产生产生活性氧种，随后激活组织因子，凝血级联的引发剂。凝血级联的诱导产生缺血莱离子是病理学相关的临床搏3。
行程具有复杂的病理生理由于许多不同的细胞类型，包括神经元，神经胶质细胞，内皮细胞和免疫系统的相互作用。选择最好的技术来研究特定的细胞过程需要多重考虑。实验技术大致可分为三类： 在体外 ， 体内和在计算机芯片与每一个具有优点和缺点体外研究有除去它们的天然环境中的细胞的主要缺点，因此可能无法再现见于一个完整的影响，。活的动物， 在体内技术提供对疾病状态具有增加的平移意义增强实验复制。 在硅片 ，一般是指一种疾病或细胞过程的计算机建模，并且同时日益用来研究对考试潜在的药物相互作用PLE，收集的任何信息仍然必须在活细胞或组织测试。
中风在实验室环境下的理想模式应该表现出类似的病理特征为那些出现在人群中。虽然有中风的人群中常见的生理特点，也有因受伤经历了种类繁多的差异。中风人群中发生的小的或大血管阻塞，出血性病变，和动脉 – 动脉或心栓塞导致在不同的梗死体积，以及在有关每一病理学机制的差异。利用动物中风模型的优点在于，模拟人类中风的特征再现的梗塞的产生。最常见的动物卒中模型包括动脉闭塞使用：大脑中动脉闭塞（栓塞血管内或长丝的方法），该模型远端缺血和photothrombosis模型。其优点是每个模型的D-缺点已在别处综述（参见图4和5）。全球局部缺血模型（MCAO），而相对容易地进行比是局灶性卒中模型不太相关人类中风。此外，这些方法都是在诱导可重复的脑梗塞病灶高度可变。该photothrombosis模型是高度可再现的，只要实验者控制他们的实验以及，提供了明显的优势MCAO模型。然而，由于微脉管损伤的模型已经描述，以显示一个最小的缺血半暗带，其中细胞被认为是挽救6,7的区域。此外，血管性水肿和细胞毒性水肿形成也可诱导的成像区域的下面照射。尽管有这些限制的技术中提供了新的认识以下中风8，9，10，11的许多生理过程。
Protocol
Representative Results
Discussion
翻译实验中风的病理生理学从动物到人类应用的能力一直饱受失败。然而，使用动物模型，如photothrombosis模式，允许中风病理生理学认识的提高和新的治疗方法提供神经保护下面中风的探索。皮质小招的光化学模型制作microinfarctions是临床相关的亚临床或“沉默”中风13-15，因而具有较高的患病率和影响约4％的美国人口（约11万人），每年16。无声中风不具有经典卒中症状存在于较?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Funding for this work was provided by: AG007218 and NIH F32 AG031606.
Images were generated in the Core Optical Imaging Facility, which is supported by UTHSCSA, NIH-NCI P30 CA54174 (CTRC at UTHSCSA) and NIH-NIA P01AG19316.
Materials
| Reagents | |||
| Rose Bengal | Sigma | 330000 | |
| Isoflurane Anesthetic | MWI Veterinary Supply | 088-076 | |
| Vetbond | 1469SB | 1469SB | |
| aCSF | 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose and 26 mM NaHCO3 (pH 7.4). | ||
| [header] | |||
| Equipment | |||
| Dissecting Scissors | Bioindustrial Products | 500-410 | |
| Operating scissors 14 cm | Bioindustrial Products | 12-055 | |
| Forceps Dumont High Tech #5 style, straight | Bioindustrial Products | TWZ-301.22 | |
| LabJack 132X80 | Optosigma Co | 123-6670 | |
| Platform for Labjack 8X 8 | Optosigma Co | 145-1110 | |
| Ear bar holder from stereotaxic setup | Stoelting/Cyborg | 51654 | |
| Dispomed Labvent Rodent anesthesia machine | DRE, Inc. | 15001 | |
| Tech IV Isoflurane vaporizer | DRE, Inc. | 34001 | |
| F Air Canister | DRE, Inc | 80120 | |
| Bain circuit breathing tube | DRE, Inc | 86111B | |
| Rodent adapter for bain tube | DRE, Inc | 891000 | |
| O2 regulator for oxygen tanks | DRE, Inc | CE001E | |
| Rodent induction chamber | DRE, Inc | 15004C | |
| Ethicon Silk 6-0; 18 in with P-3 needle | Suture Express | 1639G | |
| Objective inverter Optical Adapter | LSM technologies | ||
| Foredom drill Dual voltage 110/120 | Foredom | 134.53 |
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