This work was supported by a grant from the Institute of Medical System Engineering (iMSE) &#38; GIST-Caltech Collaborative Fund (K04592) from GIST and by the Basic Science Research Program through NRF of Korea funded by the Ministry of Science, ICT and future Planning (NRF-2013R1A2A2A01067890).

All procedures were conducted according to the institutional guidelines of Gwangju Institute of Science and Technology (GIST), and all procedures were approved by the Institutional Animal Care and Use Committee at GIST.
1. Pre-lesioning Steps
<ol>
	<li>Identification of the Forelimb Area in the Internal Capsule using AAV-GFP
	<ol>
		<li>House and handle Sprague Dawley rats (~400 g, 11 - 13 weeks) in compliance with institutional and national guidelines.</li>
		<li>Sterilize all surgical tools an...

<ol>
	<li>Roger, V. L., 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--2012+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2012 update: a report from the American Heart Association.</a> Circulation. 125, 2-220 (2012).</li><li>Debette, S., Markus, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+importance+of+white+matter+hyperintensities+on+brain+magnetic+resonance+imaging:+systematic+review+and+meta-analysis.">The clinical importance of white matter hyperintensities on brain magnetic resonance imaging: systematic review and meta-analysis.</a> Bmj. 341, 3666 (2010).</li><li>Zhang, K., Sejnowski, 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+universal+scaling+law+between+gray+matter+and+white+matter+of+cerebral+cortex.">A universal scaling law between gray matter and white matter of cerebral cortex.</a> PNAS. 97 (10), 5621-5626 (2000).</li><li>Kim, H. 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=A+rat+model+of+photothrombotic+capsular+infarct+with+a+marked+motor+deficit:+a+behavioral,+histologic,+and+microPET+study.">A rat model of photothrombotic capsular infarct with a marked motor deficit: a behavioral, histologic, and microPET study.</a> J Cereb Blood Flow Metab. 34 (4), 683-689 (2014).</li><li>Kleim, J. A., Boychuk, J. A., Adkins, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+models+of+upper+extremity+impairment+in+stroke.">Rat models of upper extremity impairment in stroke.</a> ILAR J. 48 (4), 374-384 (2007).</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 (2), 206-211 (2006).</li><li>He, Z., 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=Definition+of+the+anterior+choroidal+artery+territory+in+rats+using+intraluminal+occluding+technique.">Definition of the anterior choroidal artery territory in rats using intraluminal occluding technique.</a> J Neurol Sci. 182 (1), 16-28 (2000).</li><li>Tanaka, 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=Experimental+model+of+lacunar+infarction+in+the+gyrencephalic+brain+of+the+miniature+pig:+neurological+assessment+and+histological,+immunohistochemical,+and+physiological+evaluation+of+dynamic+corticospinal+tract+deformation.">Experimental model of lacunar infarction in the gyrencephalic brain of the miniature pig: neurological assessment and histological, immunohistochemical, and physiological evaluation of dynamic corticospinal tract deformation.</a> Stroke. 39 (1), 205-212 (2008).</li><li>Shibata, M., Ohtani, R., Ihara, M., Tomimoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White+matter+lesions+and+glial+activation+in+a+novel+mouse+model+of+chronic+cerebral+hypoperfusion.">White matter lesions and glial activation in a novel mouse model of chronic cerebral hypoperfusion.</a> Stroke. 35 (11), 2598-2603 (2004).</li><li>Watson, B. D., Dietrich, W. D., Busto, R., Wachtel, M. S., Ginsberg, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+reproducible+brain+infarction+by+photochemically+initiated+thrombosis.">Induction of reproducible brain infarction by photochemically initiated thrombosis.</a> Ann Neurol. 17 (5), 497-504 (1985).</li><li>Kuroiwa, T., 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=Development+of+a+rat+model+of+photothrombotic+ischemia+and+infarction+within+the+caudoputamen.">Development of a rat model of photothrombotic ischemia and infarction within the caudoputamen.</a> Stroke. 40 (1), 248-253 (2009).</li><li>Bashkatov, A. N., Genina, E. A., Tuchin, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of biomedical optics. 83, (2011).</li><li>Yizhar, O., Fenno, L. E., Davidson, T. J., Mogri, M., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics+in+neural+systems.">Optogenetics in neural systems.</a> Neuron. 71 (1), 9-34 (2011).</li><li>Whishaw, I. Q., Whishaw, P., Gorny, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+skilled+forelimb+reaching+in+the+rat:+a+movement+rating+scale.">The structure of skilled forelimb reaching in the rat: a movement rating scale.</a> J Vis Exp. (18), e816 (2008).</li><li>Jang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+corticospinal+tract+location+at+corona+radiata+and+posterior+limb+of+the+internal+capsule+in+human+brain.">A review of corticospinal tract location at corona radiata and posterior limb of the internal capsule in human brain.</a> NeuroRehabilitation. 24 (3), 279-283 (2009).</li><li>Kim, D., 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=Longitudinal+changes+in+resting-state+brain+activity+in+a+capsular+infarct+model.">Longitudinal changes in resting-state brain activity in a capsular infarct model.</a> J Cereb Blood Flow Metab. 35 (1), 11-119 (2014).</li><li>Blasi, F., Whalen, M. J., Ayata, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lasting+pure-motor+deficits+after+focal+posterior+internal+capsule+white-matter+infarcts+in+rats.">Lasting pure-motor deficits after focal posterior internal capsule white-matter infarcts in rats.</a> J Cereb Blood Flow Metab. 35 (6), 977-984 (2015).</li><li>Metz, G. A., Antonow-Schlorke, I., Witte, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+improvements+after+focal+cortical+ischemia+in+adult+rats+are+mediated+by+compensatory+mechanisms.">Motor improvements after focal cortical ischemia in adult rats are mediated by compensatory mechanisms.</a> Behavioural brain research. 162 (1), 71-82 (2005).</li></ol>

The authors have nothing to disclose.

The capsular infarct model presented here demonstrates a targeted lesion with marked and persistent motor impairment in forelimb function. Previous models of subcortical capsular stroke have demonstrated an insufficient degree of motor impairment and a rapid recovery process6,8,9 . In this sense, this model resembles the clinical capsular infarct cases which exhibit long-term functional impairment.
The most critical steps in the development of a circumscribed capsular infarct model ...

Until recently, the &#34;gray matter stroke (GMS) models&#34; have been exclusively used to understand the pathophysiology of stroke and to guide the development of new treatments. However, there has been an increasing prevalence of stroke that affects subcortical white matter in elderly individuals, which constitutes 15 - 25% of all strokes1,2. Numerous studies have been conducted regarding stroke using GMS models, whereas there are few studies that have used white matter stroke (WMS) models. White matter in rodents is substantially less than the white matter in humans or primates. Consequently, it is more difficult to selectively access and destroy the target regions in the white matter3. Additionally, no efficient tools have been developed to date to selectively destroy the planned extent of targeted white matter. Therefore, there has been lack of appropriate models for study of white matter strokes.
Animal stroke models are often used to monitor the progress of motor recovery for development of new rehabilitative and therapeutic methods. It is ideal to utilize an animal model that exhibits a long-term neurological deficit concordant with the anatomical alterations demonstrated in human stroke4,5. In this regard, rapid recovery of the motor deficit and wide involvement of the brain following infarct lesioning may not be realistic in the pursuit of stroke research. Previous capsular infarct models have been made by the occlusion of the internal carotid or anterior choroidal arteries and diffusion of endothelin-1 (ET-1) into the internal capsule6-9. Nonetheless, artery occlusion requires careful dissection of arteries, but it produces a wide area of infarct lesion, including the internal capsule, without persistent behavioral deficits. Moreover, ET-1 was not diffused to completely destroy the posterior limb of the internal capsule, and hence less marked or persisting behavioral deficit.
A photothrombotic infarction model has been widely used to generate various types of infarct lesions in the cortex and subcortical structures10. The technique include intravenous administration followed by focal illumination, which leads to platelet aggregation in the small vessels and generation of infarct lesions10. Photothrombotic technique has been extensively used to create GMS lesions, whereas it has rarely been used to generate WMS lesions5,11. For this technique, a combination of Rose Bengal dye and light irradiation has been demonstrated to be useful in destruction of the target structure, causing corresponding functional deficits. The key element of the photothrombotic technique is light irradiation, because it determines the size of infarct lesions. Light irradiation results in different effects on gray matter and white matter, because the scattering of light is more than 4 times higher in white matter compared with gray matter12; Accordingly, if the light intensity has a sufficiently low irradiance (&#60;1,140 mW/mm2), one can limit the extension to which photothrombotic lesion affect the extent to the white matter (i.e., internal capsule). For example, light of higher energy can induce infarcts in both gray and white matter, yet lower energy light may induce photothrombosis only in white matter. Furthermore, the penetration of light energy was very limited. Approximately 99% of light energy was lost beyond 1 mm from the source of light13. Therefore, it is expected that accurately targeted, lower energy light induces photothrombosis only in the white matter with a minimal encroachment of the neighboring gray matter.
Here, we describe a novel method to create infarct lesions in the forelimb area of the internal capsule in rodents. We describe the method of identification of the forelimb area in the internal capsule, the technology of light irradiation, including the adjustment and delivery of light, and the generation of an infarct lesion. We also describe behavioral testing used to evaluate the completeness of the capsular modeling.

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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.

&#160; The method presented here is intended to create a circumscribed capsular infarct with a persistent motor deficit. Therefore, it is critical to correctly determine the target within the internal capsule in the pre-surgery step. The somatotopic mapping of pyramidal fibers in the internal capsule has not been settled to date. To correctly identify the target within the internal capsule, the forelimb area must be delineated. An injection of AAV-GFP into the forelimb area of the motor c...

Watch this Scientific Journal Video about Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique at JoVE.com

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

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 ...

The overall goal of this technique is to create a circumscribed capsular infarct model with a persistent motor impairment using photothrombotic technique. Before creating the capsular infarct, it is necessary to identify the forelimb area in the internal capsule by introducing an adeno-associated virus expressing GFP to the forelimb area of the motor cortex. Begin by placing an anesthetized rat into a stereotaxic frame.Then shave the head of the rat and sterilize the scalp using 70%alcohol and povidone and iodine solution. Inject 2%lidocaine hydro chloride under the scalp in the area of the intended scalp incision to reduce intra operative pain. Apply ophthalmic ointment to prevent drying of the eyes.And place a sterile drape over the animal. After making a mid line incision of two centimeters in the scalp, position the stereotaxic arm to identify the forelimb area of the motor cortex according to the stereotaxic coordinates. Drill an entry hole at this position.Move the needle to the drilled hole and lower the needle one millimeter through the dura and into the motor cortex. Use a high precision micro pump to slowly inject one micro liter of virus. Leave the needle in place for an additional 10 minutes to prevent back flow up the needle tract.Once 10 minutes have elapsed, slowly withdraw the needle. After cleaning the operative site with saline irrigation, secure the wound with 3-0 nylon suture. Then release the rat from the stereotaxic frame and transfer it to a recovery chamber for monitoring.Administer analgesic for post operative pain control. After two to three weeks, sacrifice the rat according to approved methods and section the cryoprotected brain. After staining for GFP, observe the AAV-GFP transduced axons in the internal capsule with a fluorescent microscope.Compare the locations of transduced axons as seen on the fluorescent image with the rat brain atlas to accurately determine the stereotaxic coordinates of the transduced axons. To prepare the optical neural interface, first use a cutting drill to cut approximately four centimeters of a 27 gauge spinal needle with a stylet inside. Then strip 10 centimeters of the jacket of an optical fiber.Insert the un-jacketed piece of the optical fiber into a metal tube. And clamp the tube around the fiber. Then use a presser to clamp the lower 1/2 of the metal tube twice.The metal tube fills the space between the optical fiber and the hub of the spinal needle. Apply heat curable epoxy on the optical fiber. Apply additional epoxy to the empty space in the hub.And insert the optical fiber into the spinal needle. Cure the epoxy for 20 minutes at 100 degrees Celsius for stable fixation. Once cured, cleave the optical fiber that protrudes out of the spinal needle and then use diamond lapping sheets to polish the optical fiber at the tip of the spinal needle.Lastly, connect the FCPC connector part of the patch cord to the coupler of the green laser system. Then use a digital optical power and energy meter to measure the light intensity from the tip of the optical fiber to ensure accurate emission of light. Place a heat pad under the body of the animal to maintain body temperature at 37.5 degrees Celsius thought the surgery.Begin by anesthetizing and preparing the rat for surgery as before. After making a mid line incision of two centimeters, adjust the height of the nose clamp until the Bregma and Lambda are aligned at the same level. The correct alignment is critical to correctly target the internal capsule.Position the stereotaxic arm to locate the site for photothrombosis. Then drill a two millimeter hole at this point. Next, polish and clean the optical fiber tip of the ONI, then fix it to the stereotaxic frame without bending.Check the tip of the ONI to ensure it is clean and intact. Measure the laser intensity from the tip of the optical fiber prior to the insertion of the optical interface to the target site of the rat brain. Adjust the laser intensity to 3.5 milliwatts at the tip of the optical fiber.Now, lower the ONI into the target area of the internal capsule using the dorsoventral coordinate determined in the first section of the protocol. Then, inject two milligrams per kilogram of Rose Bengal through the tail vein and then start a countdown timer set to one minute. When the minute has elapsed, turn on the 532 nano-meter green laser for 90 seconds to create the infarct.After a radiation, gently remove the ONI from the brain. After cleaning the operative site, secure the wound with 3-0 nylon suture. Then remove the rat from the stereotaxic frame and transfer it to a recovery chamber.This image shows the extent of infarct lesions across varying intensities of laser light from two milliwatts to five milliwatts two weeks after photothrombotic lesioning. Arrows indicate the infarct lesion. The optical light intensity is considered to be between three milliwatts and four milliwatts in this experimental setting.The next two images show the microscopic appearance of the capsular infarct three weeks after photothrombosis. In this coronal section of the rat brain, the arrow heads indicate the tract of the needle containing the optical fiber in the thalamus and up to the internal capsule. These serial nestled stained sections show the extent of the infarct lesion in the internal capsule.The arrows indicate the infarct lesion.

circumscribed-capsular-infarct-modeling-using-photothrombotic

Research

JoVE Journal

Medicine

7.8K Görüntüleme.  Gwangju Institute of Science and Technology. The overall goal of this technique is to create a circumscribed capsular infarct model with a persistent motor impairment using photothrombotic technique. Before creating the capsular infarct, it is necessary to identify the forelimb area in the internal capsule by introducing an adeno-associated virus expressing GFP to the forelimb area of the motor cortex. Begin by placing an anesthetized rat into a stereotaxic frame.Then shave the head of the rat and sterilize the scalp using 70%alc...