A Stroke Versátil Modelo murino de Subcortical Branco Matéria para o Estudo da axonal Degeneration e branco Matéria Neurobiology
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
Acidente vascular cerebral que afecte contas de matéria branca para até 25% das apresentações clínicas acidente vascular cerebral, ocorre silenciosamente em taxas que podem ser 5-10 vezes maior, e contribui significativamente para o desenvolvimento de demência vascular. Alguns modelos de acidente vascular cerebral focal substância branca existir e esta falta de modelos apropriados tem dificultado a compreensão dos mecanismos neurobiológicos envolvidos na resposta a uma lesão e reparação após este tipo de acidente vascular cerebral. A principal limitação de outros modelos de acidente vascular cerebral subcorticais é que eles não focalmente restringir o enfarte e a substância branca ou foram validados principalmente em espécies não murinas. Isto limita a capacidade de aplicar a grande variedade de ferramentas de investigação para estudar o murino de acidente vascular cerebral neurobiologia substância branca. Aqui apresenta-se uma metodologia para a produção fiável de um acidente vascular cerebral focal em matéria branca murino utilizando uma injecção local de um inibidor de eNOS irreversível. Apresentamos também diversas variações no protocolo geral, incluindo dois estereotáxica únicavariações, traçando retrógrada neuronal, bem como de rotulagem tecido fresco e dissecação que expandir muito as potenciais aplicações desta técnica. Estas variações permitem múltiplas abordagens para analisar os efeitos neurobiológicos desta forma comum e pouco estudado de acidente vascular cerebral.
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
Um número de modelos anteriores de acidente vascular cerebral subcorticais tenham sido descritos incluindo injecções focais de endotelina-1 para a cápsula interna, matéria branca subcortical e corpo estriado do rato 6,15 12-14 e rato. Modelos mais recentes de pequenos acidentes vasculares cerebrais focais têm utilizado a injeção microembolias colesterol na artéria carótida 16 e oclusão photothrombotic de um único arteríolas penetrantes 17. Cada um destes modelos t…
Disclosures
The authors have nothing to disclose.
Acknowledgements
SN e MDD receberam apoio do NIH K08 NS083740 e do Departamento de Neurologia UCLA. AJG reconhece o apoio do Dr. Miriam e Sheldon G. Adelson Fundação de Pesquisa Médica e da L. Hillblom Fundação Larry. KLN agradece o apoio da Stroke Center American Heart Association 14BFSC17760005 ASA-Bugher. ILL, EGS e STC foram apoiados pelo NIH R01 NS071481. JDH reconhece o apoio do 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 |
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