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 height="21" style="height:21px;">Isoflurane</td>
			<td>Phoenix Pharmaceutical, Inc.</td>
			<td>NDC 57319-559-06</td>
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			<td>World Precision Instruments</td>
			<td>50-821-807</td>
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			<td>Parker Instrumentation</td>
			<td>Picospritzer II</td>
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			<td height="21" style="height:21px;">Stereotactic setup</td>
			<td>Kent Scientific</td>
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			<td>Olympus</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>
			<td>19007-05</td>
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			<td height="21" style="height:21px;">Vetbond</td>
			<td>3M</td>
			<td>1469SB&#160;</td>
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			<td height="21" style="height:21px;">Marcaine</td>
			<td>HOSPIRA</td>
			<td>NDC 0409-1610-50</td>
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			<td height="21" style="height:21px;">Trimethoprim-Sulfamethaxole</td>
			<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>
			<td></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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		<tr height="21">
			<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>
			<td></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 ...

The overall goal of this procedure is to accurately locate a small stroke within murine white matter to model this common form of subcortical white matter stroke. This method can help answer key questions in the stroke field, such as mechanisms of axonal injury, neuronal response to subcortical stroke, and how the cellular elements of white matter respond during both the acute and repair phases of stroke. The main advantage of this technique is that it can be used to precisely localize a stroke to the white matter, and can be used with a wide variety of murine mouse models.Generally, individuals new to this method will struggle, because of the small amount of white matter leading to the mislocalization of the stroke. Minor adjustments to the injection angle or stereotactic coordinates for each strain of mouse, or stereotactic setup may be required. Begin by first affixing a pulled glass pipette to tubing attached to a vacuum line.Insert the pulled end into the L-Nio solution, or L-Nio plus fluorescent tracer. Start the vacuum, and apply suction until at least two millimeters of the 0.5 millimeter diameter portion of the pipette is filled. Put the filled pipette to one side.Then place the mouse into a stereotactic apparatus equipped with a stereotactic microscope. After anesthetizing and prepping the mouse for surgery according to approved procedures, check the depth of anesthesia with a toe pinch. There should be no response.Next, adjust the injection arm to 36 degrees. Affix a pulled glass pipette holder to the distal end of a low volume pressure injection system, and attach it to the injection arm. After making a 1.5 centimeter midline scalp incision to expose the skull, dry the surface skull with a cotton swab.Then, while looking through a stereotactic microscope, at one to three X magnification, use a micropoint tool to remove any overlying periosteal tissue. Mark bregma with a fine-point marker. Then use a surgical drill equipped with a fine-tipped surgical drill bit to drill a two millimeter elliptical craniotomy, beginning posteriorly at bregma, and extending anteriorly just left of the midline.Remove bone fragments and overlying soft tissue so that the cerebral cortex can be visualized. Keep the cortical surface moist by intermittently applying drops of sterile saline. Next, affix the filled pipette to the injector arm.Align the distal end of the pipette with bregma, and zero the stereotactic coordinates. Advance the pipette to the first injection point using the anterior, posterior, and medial-lateral coordinates listed in table one. Then, advance the pipette to the cortical surface, and zero the dorsal-ventral measurement.Slowly lower the pipette to the dorsal-ventral coordinates. Ensure that the stereotactic microscope magnification is set to three X, and that the calibrated reticle can be clearly viewed. View the angled pipette from the side so that the air fluid meniscus has a sagittal view.The meniscus should appear in the same focal plane of both the inner and outer wall of the pipette. Now, using a local pressure injection system set at 20 PSI for 20 millisecond pulses, inject 100 nanoliters of L-Nio into the brain. After injecting the total volume, wait five minutes to prevent reflux up the pipette track.Then, withdraw the pipette, and repeat the injection procedure at the second and third set of coordinates provided in table one. After the final injection, remove the pipette and place enough bone wax to fill the craniotomy site. Finally, approximate the edges of the scalp wound, and bind with dermal adhesive.After administering postoperative analgesia, place the animal in a clean cage. Supply postoperative antibiotics in the drinking water for five days, or until sacrifice if less than five days. After sacrificing and decapitating the mouse according to approved procedures, use sterile scissors to open the skull posteriorly, and then use a spatula to gently remove the overlying skull.Next, insert a sterile four millimeter spatula at the front of the brain to sever the olfactory bulb and optic nerves. Then, gently lift the brain out of the calvarium and place into ice cold dissection buffer. Using a brain block and sterile razor blades, cut two to three millimeter slices containing the affected region, and place into cold dissection buffer.Under a dissecting microscope, identify the white matter underlying the motor cortex in the injected hemisphere. At earlier post-stroke intervals, injection of L-Nio mixed with common laboratory dyes, such as fast green, can allow visual identification of the stroke. At longer post-stroke intervals, the region may be visually identified by focal necrosis and myelin pallor.Once identified, use a scalpel to carefully dissect the region of white matter containing the stroke. Remove overlying cortex and underlying striatum, as desired. As shown here, immunofluorescent labeling for neural filaments, shown in red, demonstrates the degree of axonal loss seven days after stroke, using the medial approach.Using the posterior angled approach, the white matter stroke lesion is targeted just above the lateral ventricle and shows intense microglial reactivity, as detected by immunolabeling for IBA-1. Two astrocyte intermediate filament markers, vimentin, shown in red, and glial fibrillary acidic protein, shown in green, reveal changes in morphology of white matter astrocytes after stroke. Coinjection of fluorescent dextran amine at the time of stroke induction allows identification of individual neurons with axons injured by stroke.Most of the labeling occurs in axons within layer five and six neurons in primary sensorimotor cortices overlying the stroke. This image represents seven days after stroke. Once mastered, this technique can be done in 45 minutes, if it is performed properly.After watching this video, you should have a good understanding of how to produce focal white matter stroke in a mouse, and prepare the brain for a variety of downstream processing, useful to answer important questions in stroke and neuro repair.

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