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 to white matter.
1. White Matter Stroke Induction - Medial Angled Approach
<ol>
	<li>Begin by preparing a pulled glass pipette using 0.5 mm capillary tube such that the distal diameter is between 15-25 &#181;m 11.</li>
	<li>Prepare a sterile 10 &#181;l aliquot of L-Nio (N(5)-(1)-iminoethyl-L-ornithine HCl) at 27.4 mg/ml (130 &#181;M) in sterile 0.9% normal saline.</li>
	<li>Pre-fill the pulled glass pipette with a small volume of L-Nio (2-5 &#181;l) by affixing the glass pipette to tubing connected to a vacuum line. Lay the pipette flat on the bench top and insert the pulled end into the L-Nio solution.
	<ol>
		<li>Apply the vacuum until at least 2 mm of the 0.5 mm portion of the pipette is filled. Turn off the vacuum and withdraw the pipette. Place it aside until Step 1.12.</li>
	</ol>
	</li>
	<li>Place the mouse in an induction chamber and induce anesthesia of the mouse using standard 32% isoflurane flowed through a vaporizer (5 L/min inhaled with 5 L/min oxygen and 0.5 L/min N2) for 1 min or until deeply anesthesized. Transfer the mouse to a stereotactic apparatus equipped with a stereotactic microscope. Provide maintenance anesthesia using 32% isoflurane flowed through a vaporizer (2 L/min inhaled with 5 L/min oxygen and 0.5 L/min N2) and a nose cone. Check depth of anesthesia with a toe pinch.&#160;</li>
	<li>Adjust the injection arm to 36&#176;.</li>
	<li>Affix a pulled glass pipette holder to the distal end of a low volume pressure injection system and attach it to the injection arm of the stereotactic setup.</li>
	<li>Coat the anesthetized animal&#39;s whiskers with petroleum jelly and place artificial tear ointment over both eyes. Prepare a sterile surgical field by placing a sterile drape over the animal&#39;s head with a 5-10&#160;cm opening over the head.&#160;Prepare an aspetic surgical surface by shaving the fur overlying the skull. Clean the scalp with alternating betadine and 70% alcohol swabs.</li>
	<li>Make a 1.5 cm midline scalp incision with sterile fine scissors to expose the skull surface. Dry the skull with a sterile cotton swab and using a stereotactic microscope at 1-3X magnification, remove any overlying periosteal tissue using a sterile micro point tool.</li>
	<li>Mark the Bregma as a reference point using a fine point marker.</li>
	<li>Drill a 2 mm ellipitical craniotomy using a sterile fine stip surgical drill bit&#160;beginning posteriorly at the Bregma and extending anteriorly just left of the midline. Remove bone fragments and overlying soft tissue so that the cerebral cortex can be visualized.</li>
	<li>Keep the surgical field and cortical surface moist by intermittently applying drops of sterile saline.</li>
	<li>Affix a pulled glass pipette to the injector arm of the stereotactic apparatus. Align the distal end of the pipette with the Bregma and zero the stereotactic coordinates.</li>
	<li>Advance the pipette to the first anterior/posterior (A/P) and medial/lateral (M/L) coordinates provided in Table 1.</li>
	<li>Advance the pipette to the cortical surface and zero the dorsal/ventral (D/V) measurement.</li>
	<li>Slowly pass the pipette into the brain until reaching the first D/V coordinate in Table 1.</li>
	<li>Using a low volume pressure injection system set at 20 psi for 20 msec pulses, inject 100 nl of L-Nio into the brain and wait 5 min to prevent reflux up the pipette track.
	<ol>
		<li>Use a calibrated reticle in the eyepiece of the stereotactic microscope and a magnification of 3X.</li>
		<li>Accordingly, displace a total of 0.100 mm3 (0.5 mm length in a 0.5 mm diameter pipette, corresponding to 100 nl) from the pulled glass pipette for each set of coordinates. By using an reticule, measure and standardize since each set up varies depending on the magnification and scales used.</li>
		<li>For accurate volume measurement during each injection, approach the angled pipette with the microscope 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.</li>
	</ol>
	</li>
	<li>Slowly withdraw the pipette and repeat steps 1.13-1.16.3 at the second and third set of coordinates provided in Table 1.</li>
	<li>After the final injection, remove the pipette and place enough bone wax to fill the craniotomy site. Approximate the edges of the scalp wound and bind with dermal adhesive.</li>
	<li>Inject 0.1 ml of 0.5% Marcaine into the wound margins using a sterile 30 G needle to prevent&#160;to prevent local pain associated with the scalp incision.</li>
	<li>Return the animal to housing and supply post-operative antibiotics (0.48 mg/ml trimethoprim-sulfamethoxazole, or&#160;0.5 mg/ml&#160;Levofloxacin) in the drinking water for 5 days.</li>
</ol>
2. White Matter Stroke Induction - Posterior Angled Approach
<ol>
	<li>Perform steps 1.1-1.12 as in the medial angled approach protocol, except adjust the injection arm of the stereotactic setup to 45 degrees oriented anterior to posterior.</li>
	<li>Advance the pipette to the first A/P and M/L coordinates provided in Table 2.</li>
	<li>Complete remaining steps 1.14-1.20 as in the lateral angled approach protocol.</li>
</ol>
3. Retrograde Neuronal Labeling
<ol>
	<li>Prepare a sterile 10 &#181;l aliquot of L-Nio at 54.8 mg/ml in 0.9% normal saline.</li>
	<li>Prepare a sterile aliquot of 20% Fluororuby (or 20% biotinylated dextran amine or 2% Fluorogold) in 0.9% normal saline.</li>
	<li>Dilute together 1:1 for final concentrations of 27.4 mg/ml L-Nio and 10% Fluororuby.</li>
	<li>Perform the stroke protocol as above in steps 1.3-1.23.</li>
	<li>Visualize the natively fluorescent tracer in tissue section by perfusion fixation, cryosectioning and microscopy as previously described8.</li>
</ol>
4. Tissue Processing for Immunofluorescence
<ol>
	<li>At an appropriate post-stroke interval ranging from 3 hr to 14 days after stroke, euthanize mice via isoflurane overdose or local IACUC approved procedure.</li>
	<li>Open the thoracic cavity using angled scissors and insert a 23 G butterfly needle into the left ventricle.</li>
	<li>Place a small cut in the right atrium using fine scissors to allow an outflow track for the perfusion fluid.</li>
	<li>Transcardially perfuse with 30-40 ml of cold phosphate-buffered saline followed by 30-40 ml of cold 4% paraformaldehyde at a rate of 10 ml/min at RT.</li>
	<li>Decapitate the mouse and remove the brain using sterile scissors to open the skull posteriorly and then gently remove the overlying skull with a spatula, place the brain into cold 4% PFA for 24 hr, and then transfer to 30% sucrose in PBS for 48 hr.</li>
	<li>Prepare forty micron floating sections using a cryostat and perform antibody processing as previously described 6-8. In this study, use the following antibodies: rabbit anti-neurofilament 200 (1:500 dilution); rabbit anti-vimentin (1:500); goat anti-GFAP (1:500); rabbit anti-Iba-1 (1:1,000).</li>
</ol>
5. Tissue Processing for Protein or RNA Analysis
<ol>
	<li>At an appropriate post-stroke interval ranging from 3 hr to 14 days after stroke, euthanize via isoflurane overdose or local IACUC approved procedure.</li>
	<li>Decapitate the mouse and remove the brain using sterile scissors to open the skull posteriorly and then gently remove the overlying skull with a spatula.</li>
	<li>Insert a sterile 4 mm spatula at the front of the brain to sever the olfactory bulb and optic nerves. Gently lift the brain out of the calvarium and place into ice-cold dissection buffer (1x Hank&#39;s Balanced Salt Solution, 25 mM HEPES-KOH, pH 7.4, 35 mM glucose, 4 mM sodium bicarbonate, and 0.01 mg/ml cyclohexamide).</li>
	<li>Using a brain block and sterile new razor blades, prepare 2-3 mm slabs containing the stroke and place into cold dissection buffer.</li>
	<li>Under a dissecting microscope, identify the white matter underlying motor cortex in the injected hemisphere. At longer post-stroke intervals, the region may be visually identified by focal necrosis and myelin pallor. 
	Note: At earlier post-stroke intervals, injection of L-Nio mixed with 1 &#181;l of 10% Fast Green can allow visual identification of the stroke (Figure 4A).</li>
	<li>Under guidance of a dissecting microscope and using a fresh scalpel, carefully dissect the region of white matter containing the stroke, identified by either Fast Green labeling or tissue loss. Remove overlying cortex and underlying striatum as desired.</li>
</ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics--2014+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2014 update: a report from the American Heart Association.</a> Circulation. 129, 28-292 (2014).</li><li>Saini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silent+stroke:+not+listened+to+rather+than+silent.">Silent stroke: not listened to rather than silent.</a> Stroke. 43, 3102-3104 (2012).</li><li>Koton, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+and+outcome+of+prevalent+ischemic+brain+disease+in+a+national+acute+stroke+registry.">Burden and outcome of prevalent ischemic brain disease in a national acute stroke registry.</a> Stroke. 44, 3293-3297 (2013).</li><li>Jiwa, N. S., Garrard, P., Hainsworth, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+vascular+dementia+and+vascular+cognitive+impairment:+a+systematic+review.">Experimental models of vascular dementia and vascular cognitive impairment: a systematic review.</a> J Neurochem. 115, 814-828 (2010).</li><li>Sozmen, E. G., Hinman, J. D., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+that+matter:+white+matter+stroke+models.">Models that matter: white matter stroke models.</a> Neurotherapeutics. 9, 349-358 (2012).</li><li>Sozmen, E. G., Kolekar, A., Havton, L. A., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+white+matter+stroke+model+in+the+mouse:+axonal+damage,+progenitor+responses+and+MRI+correlates.">A white matter stroke model in the mouse: axonal damage, progenitor responses and MRI correlates.</a> J Neurosci Methods. 180, 261-272 (2009).</li><li>Rosenzweig, S., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+exacerbation+of+white+matter+stroke+outcomes:+a+role+for+oxidative+damage+and+inflammatory+mediators.">Age-dependent exacerbation of white matter stroke outcomes: a role for oxidative damage and inflammatory mediators.</a> Stroke. 44, 2579-2586 (2013).</li><li>Hinman, J. D., Rasband, M. N., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+the+axon+initial+segment+after+focal+cortical+and+white+matter+stroke.">Remodeling of the axon initial segment after focal cortical and white matter stroke.</a> Stroke. 44, 182-189 (2013).</li><li>McCall, T. B., Feelisch, M., Palmer, R. M., Moncada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+N-iminoethyl-L-ornithine+as+an+irreversible+inhibitor+of+nitric+oxide+synthase+in+phagocytic+cells.">Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells.</a> Brit j pharmacol. 102, 234-238 (1991).</li><li>Gadea, A., Aguirre, A., Haydar, T. F., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelin-1+regulates+oligodendrocyte+development.">Endothelin-1 regulates oligodendrocyte development.</a> J Neurosci. 29, 10047-10062 (2009).</li><li>Dean, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+(pulling)+of+needles+for+gene+delivery+by+microinjection.">Preparation (pulling) of needles for gene delivery by microinjection.</a> CSH prot. , (2006).</li><li>Hughes, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+lesions+in+the+rat+central+nervous+system+induced+by+endothelin-1.">Focal lesions in the rat central nervous system induced by endothelin-1.</a> J. Neuropathol. Exp. Neurol. 62, 1276-1286 (2003).</li><li>Whitehead, S. N., Hachinski, V. C., Cechetto, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+a+rat+model+of+cerebral+ischemia+and+beta-amyloid+toxicity:+inflammatory+responses.">Interaction between a rat model of cerebral ischemia and beta-amyloid toxicity: inflammatory responses.</a> Stroke. 36, 107-112 (2005).</li><li>Frost, S. B., Barbay, S., Mumert, M. L., Stowe, A. M., Nudo, R. 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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.

<table><tbody><tr><td>L-N5-(1-Iminoethyl)ornithine, Dihydrochloride</td><td>Calbiochem</td><td>400600-20MG</td><td></td></tr><tr><td>Isoflurane</td><td>Phoenix Pharmaceutical, Inc.</td><td>NDC 57319-559-06</td><td></td></tr><tr><td>Capillary tubes</td><td>World Precision Instruments</td><td>50-821-807</td><td></td></tr><tr><td>Picospritzer</td><td>Parker Instrumentation</td><td>Picospritzer II</td><td></td></tr><tr><td>Stereotactic setup</td><td>Kent Scientific</td><td>KSC51725</td><td></td></tr><tr><td>Pipette puller</td><td>KOPF</td><td>Model 720</td><td></td></tr><tr><td>Stereomicroscope SZ51</td><td>Olympus</td><td>88-124</td><td></td></tr><tr><td>Fine scissors</td><td>Fine Scientific Tools</td><td>14084-08</td><td></td></tr><tr><td>Forceps</td><td>Harvard Apparatus</td><td>PY2 72-8547</td><td></td></tr><tr><td>Curved Forceps</td><td>Harvard Apparatus</td><td>PY2 72-8598</td><td></td></tr><tr><td>Blunt dissection tool</td><td>Fine Scientific Tools</td><td>10066-15</td><td></td></tr><tr><td>Drill</td><td>Dremel</td><td>8220-1/28</td><td></td></tr><tr><td>Drill bits</td><td>Fine Scientific Tools</td><td>19007-05</td><td></td></tr><tr><td>Vetbond</td><td>3M</td><td>1469SB&amp;nbsp;</td><td></td></tr><tr><td>Marcaine</td><td>HOSPIRA</td><td>NDC 0409-1610-50</td><td></td></tr><tr><td>Trimethoprim-Sulfamethaxole</td><td>STI Pharmacy</td><td>NDC 54879-007-16</td><td></td></tr><tr><td>Fluororuby</td><td>Fluorochrome Inc</td><td>30 mg</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher</td><td>O4042-500</td><td></td></tr><tr><td>Sucrose</td><td>Fisher</td><td>BP220-10</td><td></td></tr><tr><td>Cryostat</td><td>Leica CM3050 S</td><td>14047033518</td><td></td></tr><tr><td>Glass slides</td><td>Fisher</td><td>12-544-7</td><td></td></tr><tr><td>Fast Green&amp;nbsp;</td><td>Sigma</td><td>F7252-5G</td><td></td></tr><tr><td>Dissection microscope</td><td>Nikon</td><td>SMZ1500</td><td></td></tr><tr><td>23 G butterfly needle</td><td>Fisher</td><td>14-840-35</td><td></td></tr><tr><td>10x Hank&#039;s Balanced Salt Solution</td><td>Life Technologies</td><td>14065056</td><td></td></tr><tr><td>1 M HEPES-KOH, pH 7.4</td><td>Affymetrix</td><td>16924</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Cyclohexamide</td><td>Sigma</td><td>01810</td><td></td></tr></tbody></table>

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 ...

a-versatile-murine-model-subcortical-white-matter-stroke-for-study

Research

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Medicine

8.0K Views.  University of California, Los Angeles. 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.

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

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Neuroscience

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...

Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Author Spotlight: Establishing a Reliable Distal MCA Occlusion Model in Mice for Stroke Research

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...

A Needle Model for Vascular Cognitive Impairment Studies in Mice

A Mouse Model for Vascular Cognitive Impairment and Dementia Based on Needle-guided Asymmetric Bilateral Common Carotid Artery Stenosis

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue rising as the population ages, it is critical to establish a robust animal model for the disease. This paper presents a novel method of creating a mouse model of VCID that is based on asymmetric bilateral common carotid artery stenosis, which mimics human chronic cerebral hypoperfusion caused by carotid atherosclerosis.
Briefly, common carotid arteries (CCAs) are ligated to different gauge needles (32 G for the right CCA and 34 G for the left CCA) using 7-0 silk sutures followed by immediate needle removal. The remaining suture rings cause persistent blood flow reduction and long-term cognitive impairment associated with white matter injury, microinfarcts, and reactive gliosis, thus closely mimicking the pathogenesis of VCID. Importantly, in this needle model, the clinical representations do not revert with time, providing reliable long-term cognitive impairment. Moreover, the survival rate 24 weeks post surgery was 81.6%, which is higher compared to the other established models of VCID with a similar level of blood flow reduction.
Additional advantages include low material cost and compatibility with MRI to monitor brain injury in live animals since no metal is implanted. The main challenge in employing the needle model of VCID is the requirement for developing advanced surgical skills since mouse CCAs are less than 0.6 mm in diameter and are very fragile. High-quality visual representation of the surgery will thus help researchers to master this technique and advance our understanding of VCID, potentially leading to the development of novel therapeutic modalities to decrease the devastating cognitive decline associated with VCID.

The needle method of asymmetric bilateral common carotid artery stenosis is proposed to create a mouse model for vascular cognitive impairment and dementia. It results in longer-term outcomes compared to previously established models and is compatible with live MRI. Visual representation demonstrating the procedure provides guidance for mastering the surgery.

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue ...

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

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