Name: a versatile murine model of subcortical white matter stroke for the study of axonal degeneration and white matter neurobiology
Uploaded: 2016-03-17T13:00:00
Description: Watch this Scientific Journal Video about A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology at JoVE.com

SN and MDD received support from NIH K08 NS083740 and the UCLA Department of Neurology. AJG acknowledges support by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and the Larry L. Hillblom Foundation. KLN gratefully acknowledges support from the American Heart Association 14BFSC17760005 ASA-Bugher Stroke Center. ILL, EGS and STC were supported by NIH R01 NS071481. JDH acknowledges support from NIH K08 NS083740.

The use of animals in this protocol has been performed in accordance with procedures approved by the University of California Los Angeles Animal Care and Use Committee.
Note: Begin by identifying the target murine population. In prior studies, only male wild-type C57/Bl6 mice have been used, however various transgenic or knockout mice can also be used. Note that stereotactic coordinates are based on C57/Bl6 anatomy. It is recommended that each user initially verify localization of the stroke t...

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A number of prior models of subcortical stroke have been described including focal injections of endothelin-1 into the internal capsule, subcortical white matter and striatum in the rat 12-14 and mouse 6,15. More recent models of small focal strokes have utilized cholesterol microemboli injection in the carotid artery 16 and photothrombotic occlusion of a single penetrating arteriole 17. Each of these models has both advantages and disadvantages 5. The presently desc...

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.

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			<td height="21" style="height:21px;width:307px;">L-N5-(1-Iminoethyl)ornithine, Dihydrochloride</td>
			<td style="width:204px;">Calbiochem</td>
			<td style="width:133px;">400600-20MG</td>
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			<td>Phoenix Pharmaceutical, Inc.</td>
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			<td>World Precision Instruments</td>
			<td>50-821-807</td>
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			<td height="21" style="height:21px;">Stereotactic setup</td>
			<td>Kent Scientific</td>
			<td>KSC51725</td>
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			<td height="21" style="height:21px;">Pipette puller</td>
			<td>KOPF</td>
			<td>Model 720</td>
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			<td>Harvard Apparatus</td>
			<td>PY2 72-8598</td>
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			<td height="21" style="height:21px;">Blunt dissection tool</td>
			<td>Fine Scientific Tools</td>
			<td>10066-15</td>
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			<td>Fine Scientific Tools</td>
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			<td>3M</td>
			<td>1469SB&#160;</td>
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			<td>HOSPIRA</td>
			<td>NDC 0409-1610-50</td>
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			<td>STI Pharmacy</td>
			<td>NDC 54879-007-16</td>
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			<td height="21" style="height:21px;">Fluororuby</td>
			<td>Fluorochrome Inc</td>
			<td>30mg</td>
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			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Fisher</td>
			<td>O4042-500</td>
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			<td height="21" style="height:21px;">Sucrose</td>
			<td>Fisher</td>
			<td>BP220-10</td>
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			<td height="21" style="height:21px;">Cryostat</td>
			<td>Leica CM3050 S</td>
			<td>14047033518</td>
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			<td height="21" style="height:21px;">Glass slides</td>
			<td>Fisher</td>
			<td>12-544-7</td>
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			<td height="21" style="height:21px;">Fast Green&#160;</td>
			<td>Sigma</td>
			<td>F7252-5G</td>
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			<td height="21" style="height:21px;">Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ1500</td>
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			<td height="21" style="height:21px;">23 gauge butterfly needle</td>
			<td>Fisher</td>
			<td>14-840-35</td>
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			<td height="21" style="height:21px;">10X Hank&#39;s Balanced Salt Solution</td>
			<td>Life Technologies</td>
			<td>14065056</td>
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			<td height="21" style="height:21px;">1M HEPES-KOH, pH 7.4</td>
			<td>Affymetrix</td>
			<td>16924</td>
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			<td height="21" style="height:21px;">D-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
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			<td height="21" style="height:21px;">Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
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			<td height="21" style="height:21px;">Cyclohexamide</td>
			<td>Sigma</td>
			<td>01810</td>
			<td></td>
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a versatile murine model of subcortical white matter stroke for the study of axonal degeneration and white matter neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Using the model presented, the white matter underlying forelimb sensorimotor cortex can reliably be targeted. This chemically induced stroke model produces focal axonal and myelin loss, astrocytosis, and microgliosis (Figure 1), as is typically seen in human lacunar infarcts. By using three injections, a clinically useful model is established with early impairment on forelimb motor tasks 7 and a small but significant portion of brain tissue experiences ischemia...

Watch this Scientific Journal Video about A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology at JoVE.com

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

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.

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

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