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>

<ol>
	<li>Go, A. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+animal+model+of+capsular+infarct:+endothelin-1+injections+in+the+rat.">An animal model of capsular infarct: endothelin-1 injections in the rat.</a> Behav Brain Res. 169, 206-211 (2006).</li><li>Horie, N., 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=Mouse+model+of+focal+cerebral+ischemia+using+endothelin-1.">Mouse model of focal cerebral ischemia using endothelin-1.</a> J Neurosci Methods. 173, 286-290 (2008).</li><li>Wang, 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=Cognitive+deficits+and+delayed+neuronal+loss+in+a+mouse+model+of+multiple+microinfarcts.">Cognitive deficits and delayed neuronal loss in a mouse model of multiple microinfarcts.</a> Neuroscience. 32, 17948-17960 (2012).</li><li>Shih, A. Y., 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=The+smallest+stroke:+occlusion+of+one+penetrating+vessel+leads+to+infarction+and+a+cognitive+deficit.">The smallest stroke: occlusion of one penetrating vessel leads to infarction and a cognitive deficit.</a> Nat Neurosci. 16, 55-63 (2013).</li><li>Jung, K. J., 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=The+role+of+endothelin+receptor+A+during+myelination+of+developing+oligodendrocytes.">The role of endothelin receptor A during myelination of developing oligodendrocytes.</a> J Korean Med Sci. 26, 92-99 (2011).</li></ol>

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 described model produces a lesion that has a number of characteristics that mimic human lacunar infarction including axonal abnormalities and loss, myelin degradation, a focal necrotic core and a clinical deficit that is minimal and demonstrates fairly rapid recovery dependent on age 7. Targeting murine white matter allows a wide variety of genetic manipulations that can support mechanistic studies.
The critical steps of the protocol include utilizing accurate stereotactic coordinates. Because murine white matter is small in size and varies from strain to strain, revision of the stereotactic coordinates may be needed depending on the age and strain of mouse used. Control of the volume of injection is also important as this is directly correlated to the size of the infarct. Larger injections will produce larger strokes that encroach on overlying cortex and underlying striatum.
Focal injection of ET-1 using the approach described here has been reported but endothelin-1 was shown to have direct paracrine effects on oligodendrocyte differentiation and maturation 10,18 confounding the study of post-stroke white matter biology. In contrast, the L-Nio approach targets endothelial cells alone while producing an identical lesion and eliminates any confounding paracrine effects on the cells involved in injury response. L-Nio is not directly cytotoxic and the selected dose was determined by preliminary dose escalation experiments (data not shown). The posterior angled approach was developed to more precisely undercut axons from primary motor cortex producing the maximal behavioral deficit that can be attributed to white matter injury. The medial angled approach also damages motor cortex axons but extends more laterally and involves axons underlying primary sensory cortex.
The stroke lesion expands fairly rapidly over the first 24 hr. By seven days, the size of the infarct is maximal and we have not observed significant lesion growth beyond that time. Additional cellular events and axonal degeneration will occur beyond this initial stage but the infarct size as measured by the necrotic core will not change significantly in the absence of any intervention.
Co-injection of neuroanatomical tracers at the time of stroke identifies neurons experiencing ischemic axonal injury. Using either the medial or posterior angled approach, the neuronal cell bodies with damaged axonal projections remain unharmed. This creates a useful model to study the effect of ischemic axotomy on central nervous system neurons. We have utilized primarily retrograde tracers including dextran amine and fluorogold, which both show excellent uptake by stroke-damaged axons. By employing the wide variety of transgenic and knock-out mouse lines, users of this protocol can investigate the specific role of neuronal genes involved in white matter stroke injury response and repair.

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 border="0" cellpadding="0" cellspacing="0" width="725">
	<tbody>
		<tr height="21">
			<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>
			<td style="width:81px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Phoenix Pharmaceutical, Inc.</td>
			<td>NDC 57319-559-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary tubes</td>
			<td>World Precision Instruments</td>
			<td>50-821-807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Picospritzer</td>
			<td>Parker Instrumentation</td>
			<td>Picospritzer II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereotactic setup</td>
			<td>Kent Scientific</td>
			<td>KSC51725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette puller</td>
			<td>KOPF</td>
			<td>Model 720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereomicroscope SZ51</td>
			<td>Olympus</td>
			<td>88-124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine scissors</td>
			<td>Fine Scientific Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Harvard Apparatus</td>
			<td>PY2 72-8547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved Forceps</td>
			<td>Harvard Apparatus</td>
			<td>PY2 72-8598</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt dissection tool</td>
			<td>Fine Scientific Tools</td>
			<td>10066-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drill</td>
			<td>Dremel</td>
			<td>8220-1/28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drill bits</td>
			<td>Fine Scientific Tools</td>
			<td>19007-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vetbond</td>
			<td>3M</td>
			<td>1469SB&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Marcaine</td>
			<td>HOSPIRA</td>
			<td>NDC 0409-1610-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trimethoprim-Sulfamethaxole</td>
			<td>STI Pharmacy</td>
			<td>NDC 54879-007-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluororuby</td>
			<td>Fluorochrome Inc</td>
			<td>30mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Fisher</td>
			<td>O4042-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Fisher</td>
			<td>BP220-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Leica CM3050 S</td>
			<td>14047033518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass slides</td>
			<td>Fisher</td>
			<td>12-544-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green&#160;</td>
			<td>Sigma</td>
			<td>F7252-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ1500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">23 gauge butterfly needle</td>
			<td>Fisher</td>
			<td>14-840-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X Hank&#39;s Balanced Salt Solution</td>
			<td>Life Technologies</td>
			<td>14065056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1M HEPES-KOH, pH 7.4</td>
			<td>Affymetrix</td>
			<td>16924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 that immunohistochemical, immunofluorescent, and biochemical techniques are feasible and reliable at the quantitative level. At early time points (hr) after stroke induction, changes in axonal molecular organization can be detected (Figure 2). At 7 days, the stroke completes its maturation to a circumscribed area of axonal loss (Figure 1a). In our hands, this method produces a focal white matter lesion approximately 800 &#181;m in horizontal diameter and extending approximately 1 mm along the anterior-posterior axis of the corpus callosum. By 7 days, the average total infarct size is roughly 0.200 mm3 and will have an elliptical shape. We have observed approximately 10% variability in stroke size both between animals and between sections, dependent on where in the ellipse the section occurs. Further growth in the size of the infarct is rarely seen beyond 7 days.
The addition of a simultaneous injection of dextran amine results in significant neuronal labeling in layer 5 and layer 6 neuronal cell bodies that have axons projecting through the region of stroke (Figure 3). Overlying cortical neurons that are not damaged by the sham aspects of the procedure (passage of fine needle), undergo distal axonal damage and can be identified by the inclusion of a tracer with the L-Nio preparation. This approach was used to demonstrate dynamic changes in the axon initial segment after stroke 8.
While the small size of the region of tissue affected by the stroke limits the use of other common techniques such as 2,3,5-triphenyltetrazolium chloride (TTC) staining, the white matter stroke region can be identified in fresh tissue. The addition of a common dye such as Fast Green produces an identifiable region of tissue that can be dissected under a dissecting microscope (Figure 4). Once dissected this tissue can be used for protein analysis with western blot or immunoprecipitation, or for RNA isolation and analysis (Figure 4C). Through the use of various transgenic mouse lines, a variety of innovative approaches can be used to study the neurobiology after focal white matter stroke including cell fate mapping studies and laser capture microdissection (Figure 4C).
<img alt="Figure 1" src="/files/ftp_upload/53404/53404fig1.jpg" /> 
Figure 1: Focal White Matter Stroke Using Both Medial and Posterior Angled Approaches. Immunofluorescent labeling for neurofilaments (A, 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 (B, C) and shows intense microglial (B) and astrocytic reactivity (C). Two astrocyte intermediate filament markers, vimentin (red) and glial fibrillary acidic protein (GFAP, green), both reveal changes in morphology of white matter (fibrous) astrocytes after stroke (C). Scale bars = 500 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53404/53404fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53404/53404fig2.jpg" /> 
Figure 2: White Matter Stroke Alters Axonal Microdomain Organization. Within 3 hr of white matter stroke induction, axonal microdomain organization at the node, marked by beta-IV spectrin (red) and at the paranode, marked by contactin-associated protein (caspr, green), is disrupted (arrows, B). Contralateral white matter axons show regular nodal, paranodal, and juxtaparanodal organization (A and C) while the ipsilateral white matter shows nodal and paranodal elongation that is typical of axons with lost axoglial contact (B and D). Scale bar = 5 &#956;m.
<img alt="Figure 3" src="/files/ftp_upload/53404/53404fig3.jpg" /> 
Figure 3: Retrograde Neuronal Labeling with White Matter Stroke Identifies Individual Neurons with Axonal Damage. Co-injection of fluorescent dextran amine (red) 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 5 and 6 neurons in primary sensorimotor cortices overlying the stroke. Image represents 7 days after stroke. Scale bar = 500 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53404/53404fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53404/53404fig4.jpg" /> 
Figure 4: Microdissection of White Matter Stroke Lesions for Use in Biochemical and Transcriptional Assays. Co-injection of Fast Green at the time of stroke induction allows early identification of the injured region at 24 hr (A, upper panel). Immunoblotting for specific axonal proteins can be performed to demonstrate reductions in the region of stroke alone (A, lower panel). At longer time points, the region of white matter stroke (left panel) can be focally dissected without specific labeling (B). Right upper panel shows region of white matter after removal of dissected region while the lower right panel shows the dissected region of white matter containing the stroke (B). PCR for oligodendrocyte-specific genes using RNA isolated from dissected regions from the white matter (C). IL = ipsilateral; CL = contralateral; c = control; s = stroke.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">Injection</td>
			<td>Anterior/Posterior</td>
			<td>Medial/Lateral</td>
			<td>Dorsal/Ventral</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;">1</td>
			<td align="right">0.22</td>
			<td align="right">0.22</td>
			<td align="right">-2.10</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;">2</td>
			<td align="right">0.70</td>
			<td align="right">0.15</td>
			<td align="right">-2.16</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;">3</td>
			<td align="right">1.21</td>
			<td align="right">0.15</td>
			<td align="right">-2.18</td>
		</tr>
	</tbody>
</table>
Table 1: Stereotactic Coordinates for Lateral Angled Approach (in mm).
<table fo:keep-together.within-page="1">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">Injection</td>
			<td>Anterior/Posterior</td>
			<td>Medial/Lateral</td>
			<td>Dorsal/Ventral</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;">1</td>
			<td align="right">-0.75</td>
			<td align="right">-0.96</td>
			<td align="right">-2.10</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;">2</td>
			<td align="right">-1.00</td>
			<td align="right">-0.96</td>
			<td align="right">-2.05</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;">3</td>
			<td align="right">-1.25</td>
			<td align="right">-0.96</td>
			<td align="right">-2.00</td>
		</tr>
	</tbody>
</table>
Table 2: Stereotactic Coordinates for Posterior Angled Approach (in mm).

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

Universidad San Sebastian

Research

JoVE Journal

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

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

DTI of the Visual Pathway - White Matter Tracts and Cerebral Lesions

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to visual pathways (VP), WMT are not visible with classical MRI or intra-operatively with microscope. DTI will help neurosurgeons to prevent destruction of the VP while removing lesions adjacent to this WMT. We have performed DTI on fifty patients before and after surgery between March 2012 to January 2014. To navigate we used a 3DT1-weighted sequence. Additionally, we performed a T2-weighted and DTI-sequences. The parameters used were, FOV: 200 x 200 mm, slice thickness: 2 mm, and acquisition matrix: 96 x 96 yielding nearly isotropic voxels of 2 x 2 x 2 mm. Axial MRI was carried out using a 32 gradient direction and one b0-image. We used Echo-Planar-Imaging (EPI) and ASSET parallel imaging with an acceleration factor of 2 and b-value of 800 s/mm&#178;. The scanning time was less than 9 min.
The DTI-data obtained were processed using a FDA approved surgical navigation system program which uses a straightforward fiber-tracking approach known as fiber assignment by continuous tracking (FACT). This is based on the propagation of lines between regions of interest (ROI) which is defined by a physician. A maximum angle of 50, FA start value of 0.10 and ADC stop value of 0.20 mm&#178;/s were the parameters used for tractography.
There are some limitations to this technique. The limited acquisition time frame enforces trade-offs in the image quality. Another important point not to be neglected is the brain shift during surgery. As for the latter intra-operative MRI might be helpful. Furthermore the risk of false positive or false negative tracts needs to be taken into account which might compromise the final results.

Diffusion tensor imaging (DTI) was performed to try to depict the major parts of the visual pathway. The goal was to use an FDA approved standard commercial workstation which could be used for everyday routine in order to try to reduce postoperative damage of the visual pathway in the patients.

DTI is a technique that identifies white matter tracts (WMT) non-invasively in healthy and non-healthy patients using diffusion measurements. Similar to ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle screws after spinal surgery, which is a serious concern. To address this problem, diverse types of pedicle screws have been examined to identify those with good fixation strength and osseointegration in spine bone. The porcine spine is a good alternative for the human spine in the evaluation of pedicle screws due to the anatomical size, mechanical characteristics, and cost. Although several studies have reported that pedicle screws are efficient in the porcine model, no study has described detailed protocols for the evaluation of a pedicle screw using the porcine model. Here, we describe a detailed method for evaluating transpedicular screws using an in vivo porcine lumbar spine model. The technical details for anesthesia, spine surgery, and harvest provided here will facilitate with the evaluation of the transpedicular screw fixation model.

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle ...