軸索変性と白質神経生物学の研究のための皮質下白質ストロークの多彩なマウスモデル
Summary
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.
Abstract
ストローク臨床ストロークプレゼンテーションの最大25％を白質アカウントに影響を与え、5〜10倍大きくすることができる速度で静かに発生し、血管性認知症の発展に大きく貢献しています。焦点白質ストロークのいくつかのモデルが存在し、適切なモデルの欠如は、脳卒中のこのタイプの後に損傷応答と修復に関与する神経生物学のメカニズムの理解を妨げています。他の皮質下脳卒中モデルの主な制限は、彼らが限局的に白質に梗塞を制限しないか、主に非マウス種で確認されていることです。これは、白質ストロークの神経生物学を研究するために、マウス研究ツールを幅広く適用する能力を制限します。ここでは、不可逆的なeNOSの阻害剤の局所注射を使用して、マウスの白質における焦点脳卒中の信頼性の高い生産のための方法論を提示します。また、2つのユニークな定位を含む一般的なプロトコルにいくつかのバリエーションを提示しますバリエーション、大幅にこの技術の潜在的な用途を拡大する逆行性神経のトレースだけでなく、新鮮な組織標識および解剖。これらの変化は、脳卒中のこの共通とunderstudied形の神経生物学的効果を分析するための複数のアプローチを可能にします。
Introduction
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.
Protocol
Representative Results
Discussion
皮質下脳卒中の前モデルの数は、ラット12-14とマウス6,15における内部のカプセルへのエンドセリン-1の焦点注射、皮質下白質と線条体を含む、記載されています。小焦点脳卒中のより最近のモデルは、頸動脈16と、単一の貫通動脈17の光血栓閉塞中のコレステロール微小塞栓注射を利用してきました。これらのモデルのそれぞれが長所と短所5の両方を?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
SNとMDDは、NIH K08 NS083740と神経のUCLA省から支援を受けました。 AJG博士ミリアムとシェルドン・G・アデルソン医学研究財団とラリーL. Hillblom基金による支援を認めます。 KLNは感謝アメリカ心臓協会14BFSC17760005 ASA-Bugher脳卒中センターから支援を認めています。 ILL、EGSとSTCは、NIH R01 NS071481によってサポートされていました。 JDHは、NIH K08 NS083740からの支援を認めています。
Materials
| 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 |
References
- Go, A. S., et al. Heart disease and stroke statistics–2014 update: a report from the American Heart Association. Circulation. 129, 28-292 (2014).
- Saini, M., et al. Silent stroke: not listened to rather than silent. Stroke. 43, 3102-3104 (2012).
- Koton, S., et al. Burden and outcome of prevalent ischemic brain disease in a national acute stroke registry. Stroke. 44, 3293-3297 (2013).
- Jiwa, N. S., Garrard, P., Hainsworth, A. H. Experimental models of vascular dementia and vascular cognitive impairment: a systematic review. J Neurochem. 115, 814-828 (2010).
- Sozmen, E. G., Hinman, J. D., Carmichael, S. T. Models that matter: white matter stroke models. Neurotherapeutics. 9, 349-358 (2012).
- Sozmen, E. G., Kolekar, A., Havton, L. A., Carmichael, S. T. A white matter stroke model in the mouse: axonal damage, progenitor responses and MRI correlates. J Neurosci Methods. 180, 261-272 (2009).
- Rosenzweig, S., Carmichael, S. T. Age-dependent exacerbation of white matter stroke outcomes: a role for oxidative damage and inflammatory mediators. Stroke. 44, 2579-2586 (2013).
- Hinman, J. D., Rasband, M. N., Carmichael, S. T. Remodeling of the axon initial segment after focal cortical and white matter stroke. Stroke. 44, 182-189 (2013).
- McCall, T. B., Feelisch, M., Palmer, R. M., Moncada, S. Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells. Brit j pharmacol. 102, 234-238 (1991).
- Gadea, A., Aguirre, A., Haydar, T. F., Gallo, V. Endothelin-1 regulates oligodendrocyte development. J Neurosci. 29, 10047-10062 (2009).
- Dean, D. A. Preparation (pulling) of needles for gene delivery by microinjection. CSH prot. , (2006).
- Hughes, P. M., et al. Focal lesions in the rat central nervous system induced by endothelin-1. J. Neuropathol. Exp. Neurol. 62, 1276-1286 (2003).
- Whitehead, S. N., Hachinski, V. C., Cechetto, D. F. Interaction between a rat model of cerebral ischemia and beta-amyloid toxicity: inflammatory responses. Stroke. 36, 107-112 (2005).
- Frost, S. B., Barbay, S., Mumert, M. L., Stowe, A. M., Nudo, R. J. An animal model of capsular infarct: endothelin-1 injections in the rat. Behav Brain Res. 169, 206-211 (2006).
- Horie, N., et al. Mouse model of focal cerebral ischemia using endothelin-1. J Neurosci Methods. 173, 286-290 (2008).
- Wang, M., et al. Cognitive deficits and delayed neuronal loss in a mouse model of multiple microinfarcts. Neuroscience. 32, 17948-17960 (2012).
- Shih, A. Y., et al. The smallest stroke: occlusion of one penetrating vessel leads to infarction and a cognitive deficit. Nat Neurosci. 16, 55-63 (2013).
- Jung, K. J., et al. The role of endothelin receptor A during myelination of developing oligodendrocytes. J Korean Med Sci. 26, 92-99 (2011).
