Et alsidigt murin model af subkortikale White Matter Stroke for Studiet af axonal degeneration og White Matter Neurobiologi
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
Slagtilfælde rammer hvid substans udgør op til 25% af kliniske slagtilfælde præsentationer, sker lydløst ved hastigheder, der kan være 5-10 gange større, og bidrager væsentligt til udviklingen af vaskulær demens. Kun få modeller af fokal hvide substans slagtilfælde eksisterer, og denne mangel på egnede modeller har hæmmet forståelse af de neurobiologic mekanismer involveret i skade respons og reparation efter denne form for slagtilfælde. Den væsentligste begrænsning af andre subkortikale slagtilfælde modeller er, at de ikke fokalt begrænse infarktet og den hvide substans eller er primært blevet valideret i ikke-murine arter. Dette begrænser muligheden for at anvende den brede vifte af murine forsknings- værktøjer til at studere neurobiologi af hvide substans slagtilfælde. Her præsenterer vi en metode til pålidelig produktion af et samlingspunkt slagtilfælde i murine hvide substans ved hjælp af en lokal injektion af en irreversibel eNOS inhibitor. Vi præsenterer også flere variationer af den generelle protokol herunder to unikke stereotaktiskvariationer, retrograd neuronal tracing, samt frisk mærkning væv og dissektion, som i høj grad udvide de potentielle anvendelser af denne teknik. Disse variationer muliggøre flere tilgange til at analysere de neurobiologic virkningerne af denne fælles og dublerede form for slagtilfælde.
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
Et antal tidligere modeller af subkortikal slagtilfælde er blevet beskrevet herunder fokale injektioner af endothelin-1 i det indre kapsel, subkortikal hvid substans og striatum hos rotter 12-14 og mus 6,15. Nyere modeller af små fokale slagtilfælde har udnyttet kolesterol microemboli injektion i halspulsåren 16 og photothrombotic okklusion af en enkelt gennemtrængende arteriole 17. Hver af disse modeller har både fordele og ulemper 5. Den foreliggende beskrev…
Disclosures
The authors have nothing to disclose.
Acknowledgements
SN og MDD fået støtte fra NIH K08 NS083740 og UCLA Department of Neurology. AJG anerkender støtte fra Dr. Miriam og Sheldon G. Adelson Medical Research Foundation og Larry L. Hillblom Foundation. KLN taknemmeligt anerkender støtte fra American Heart Association 14BFSC17760005 ASA-Bugher Stroke Center. ILL, EGS og STC blev støttet af NIH R01 NS071481. JDH anerkender støtte fra 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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