A Corsa Versatile Modello murino di Subcortical bianco Materia per lo Studio della assonale degenerazione e bianco Materia Neurobiologia
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
Ictus colpisce conti sostanza bianca fino al 25% delle presentazioni ictus cliniche, avviene automaticamente a velocità che possono essere 5-10 volte maggiore, e contribuiscono alla sviluppo di demenza vascolare. Pochi modelli di focale ictus sostanza bianca esistono e questa mancanza di modelli appropriati ha ostacolato la comprensione dei meccanismi coinvolti nella risposta neurobiologiche danno e la riparazione dopo questo tipo di ictus. La principale limitazione di altri modelli di ictus subcorticali è che essi non focally limitano l'infarto alla materia bianca o sono stati in primo luogo convalidato in specie non murini. Questo limita la capacità di applicare l'ampia varietà di strumenti di ricerca murini per studiare la neurobiologia di bianco ictus materia. Qui presentiamo una metodologia per la produzione affidabile di un ictus focale nella materia bianca murino utilizzando una iniezione locale di un inibitore irreversibile eNOS. Presentiamo anche diverse variazioni sul protocollo generale di cui due stereotassica unicovariazioni, retrograda tracing neuronale, nonché di etichettatura tessuto fresco e sezionamento che ampliare notevolmente le potenziali applicazioni di questa tecnica. Queste variazioni permettono di molteplici approcci per analizzare gli effetti neurobiologiche di questa forma comune e poco studiato di ictus.
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
Un certo numero di modelli precedenti di ictus sottocorticale sono stati descritti tra cui iniezioni focali di endotelina-1 nella capsula interna, sostanza bianca sottocorticale e striato nel ratto 6,15 12-14 e mouse. Modelli più recenti di piccoli colpi focali hanno utilizzato il colesterolo microemboli iniezione nella carotide 16 e l'occlusione photothrombotic di un singolo arteriole penetrante 17. Ognuno di questi modelli presenta vantaggi e svantaggi 5. Il …
Disclosures
The authors have nothing to disclose.
Acknowledgements
SN e MDD hanno ricevuto sostegno dal NIH K08 NS083740 e il Dipartimento di Neurologia della UCLA. AJG riconosce sostegno da parte del Dr. Miriam e Adelson Research Foundation Sheldon G. medica e la L. Hillblom Fondazione Larry. KLN ringrazia il sostegno della Stroke Centro American Heart Association 14BFSC17760005 ASA-Bugher. ILL, EGS e STC sono stati sostenuti da NIH R01 NS071481. JDH riconosce il sostegno NIH K08 NS083740.
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. 신경과학. 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).
