Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.
中风影响白质占的临床冲程演示高达25%,在费率可能5-10倍更大默默地发生,并且显著血管性痴呆的发展有助于。存在焦白质中风的几个型号,这缺乏适当的模式阻碍了参与这类中风后损伤反应和修复neurobiologic机制的理解。其他皮层下中风模型的主要限制是,它们不灶性限制梗塞的白质或已经主要在非鼠物种得到验证。这限制应用各种各样的鼠研究工具来研究白质中风的神经生物学的能力。在这里,我们提出了一个方法,为可靠的生产使用的是不可逆的eNOS抑制剂局部注射鼠白质的焦点中风。我们还提出对一般协议,包括两个独特的立体几个变化变型中,逆行神经追踪,以及新鲜组织标记和解剖,大大扩大该技术的潜在应用。这些变化允许多种方法来分析的行程这个共同的和充分研究形式neurobiologic影响。
Stroke affecting the subcortical white matter is a common clinical entity, accounting for up to 25% of clinical strokes annually in the US 1. Ischemic damage to white matter also occurs silently at a significantly higher rate and contributes to the development of vascular dementia 2,3. Presently, patients with this form of cerebral ischemia have few, if any treatment choices. Despite the clinical importance of this disease, few clinically relevant animal models exist 4,5.
The goal of this protocol is to produce a focal ischemic lesion within the murine white matter. This murine model of human disease allows the specific study of axonal injury response to stroke and how the cellular elements of white matter, namely oligodendrocytes and astrocytes along with axons, respond to and repair after stroke.
Previous reports have described a model of subcortical white matter stroke using endothelin-1 (ET-1) 6 that is similar to the one described here. Several key changes to the experimental protocol have been made thereby the potential uses of this model have expanded 7,8. This protocol provides a reliable and modifiable strategy to produce a focal stroke within mouse brain white matter.
The major advantages of this model are the use of a chemical endothelial nitric oxide synthase (eNOS) inhibitor N(5)-(1)-iminoethyl-L-ornithine HCl (L-Nio) 9 with no known paracrine effects on cellular elements of white matter which had been a complication of models using endothelin-1 10. In addition, the stereotactic targeting of white matter in the mouse allows the use of any variety of transgenic or knockout strains, greatly expanding the available tools to determine the effect of stroke on brain white matter. Here, two variations on this technique are described and demonstrate some of the additional variations that can be utilized to enhance the understanding of axonal and white matter damage and repair after stroke.
已经描述了若干皮层下行程的前模型,包括内皮素-1的焦点注射进入内囊,皮层下白质和纹状体在大鼠12-14和鼠标6,15。更近的小焦点冲程模型已经利用在颈动脉16胆固醇微栓子注射和单个穿透动脉17的光化学闭塞。每个模型都有有利有弊5。目前描述的模型产生具有许多模仿人类腔隙性脑梗塞,包括轴突异常和损失,髓鞘退化,焦距坏死核心和临床赤字是最?…
The authors have nothing to disclose.
SN和MDD从NIH K08 NS083740和神经病学的加州大学洛杉矶分校部门的支持。 AJG承认由仪博士和谢尔登·阿德尔森G.医学研究基金会和拉里·希尔布鲁姆L.基金会的支持。九龙十分感谢美国心脏协会14BFSC17760005 ASA-Bugher卒中中心的支持。 ILL,EGS和STC是由美国国立卫生研究院R01 NS071481支持。 JDH确认来自美国国立卫生研究院K08 NS083740支持。
L-N5-(1-Iminoethyl)ornithine, Dihydrochloride | Calbiochem | 400600-20MG | |
Isoflurane | Phoenix Pharmaceutical, Inc. | NDC 57319-559-06 | |
Capillary tubes | World Precision Instruments | 50-821-807 | |
Picospritzer | Parker Instrumentation | Picospritzer II | |
Stereotactic setup | Kent Scientific | KSC51725 | |
Pipette puller | KOPF | Model 720 | |
Stereomicroscope SZ51 | Olympus | 88-124 | |
Fine scissors | Fine Scientific Tools | 14084-08 | |
Forceps | Harvard Apparatus | PY2 72-8547 | |
Curved Forceps | Harvard Apparatus | PY2 72-8598 | |
Blunt dissection tool | Fine Scientific Tools | 10066-15 | |
Drill | Dremel | 8220-1/28 | |
Drill bits | Fine Scientific Tools | 19007-05 | |
Vetbond | 3M | 1469SB | |
Marcaine | HOSPIRA | NDC 0409-1610-50 | |
Trimethoprim-Sulfamethaxole | STI Pharmacy | NDC 54879-007-16 | |
Fluororuby | Fluorochrome Inc | 30mg | |
Paraformaldehyde | Fisher | O4042-500 | |
Sucrose | Fisher | BP220-10 | |
Cryostat | Leica CM3050 S | 14047033518 | |
Glass slides | Fisher | 12-544-7 | |
Fast Green | Sigma | F7252-5G | |
Dissection microscope | Nikon | SMZ1500 | |
23 gauge butterfly needle | Fisher | 14-840-35 | |
10X Hank's Balanced Salt Solution | Life Technologies | 14065056 | |
1M HEPES-KOH, pH 7.4 | Affymetrix | 16924 | |
D-Glucose | Sigma | G8270 | |
Sodium bicarbonate | Sigma | S5761 | |
Cyclohexamide | Sigma | 01810 |