Effect of Suture Properties on Stability of Middle CerebralArtery Occlusion Evaluated by Synchrotron

Radiation AngiographyYongjing Guan, MD; Yongting Wang, PhD; Falei Yuan, MS; Haiyan Lu, MD; Yuqi Ren, PhD;

Tiqiao Xiao, PhD; Kemin Chen, MD, PhD; David A. Greenberg, MD, PhD;Kunlin Jin, MD, PhD; Guo-Yuan Yang, MD, PhD

Background and Purpose—The intraluminal suture technique for producing middle cerebral artery occlusion in rodentsis the most commonly used method for modeling focal cerebral ischemia associated with clinical ischemic stroke.Synchrotron radiation angiography may provide a novel solution to directly monitor the success of middle cerebralartery occlusion.

Methods—Twenty adult Sprague-Dawley rats for middle cerebral artery occlusion models were prepared randomly withdifferent suture head silicone coating. In vivo imaging was performed at beam line BL13W1, Shanghai SynchrotronRadiation Facility, Shanghai, China.

Results—Silicone-coated suture was superior to uncoated suture for producing consistent brain infarction. Additionally,silicone coating length was an important variable controlling the extent of the ischemic lesion: infarcts affectedpredominantly the caudate–putamen with large variability (�2 mm), both the cortex and caudate–putamen (2–3.3 mm),and most of the hemisphere, including the hypothalamus (�3.3 mm).

Conclusions—Synchrotron radiation angiography provides a useful tool to observe hemodynamic changes after middlecerebral artery occlusion, and the physical properties of suture are critical to the success of the middle cerebral arteryocclusion model. (Stroke. 2012;43:888-891.)

Key Words: angiography � middle cerebral artery occlusion � synchrotron radiation

The focal cerebral ischemia model involves the occlusionof the middle cerebral artery (MCAO) and typically

results in localized brain infarction, which recapitulates manyof the pathophysiological and histopathologic features ofstroke. The most common technique for MCAO is theintraluminal filament model.1 However, 1 limitation of thesuture model is its high variability in infarct size.2 Factors thatcontribute to such variation include differences in animalstrain and weight, method of anesthesia, blood pressure, brainand body temperature, brain vascular anatomy, suture mate-rial, duration, and site of occlusion. Physical properties of thefilament are important because it is the key factor that affectslesion volume3; however, there is no direct evidence toconfirm this principle.4,5

Regional cerebral blood flow can be measured usingautoradiography and laser Doppler flowmetry to confirmsuccessful MCAO, but neither method can dynamicallymonitor changes of cerebral blood flow in deeper tissueduring ischemia–reperfusion.6,7 Synchrotron radiation an-

giography (SRA) may represent a novel solution to directlyand dynamically monitor MCAO.8 In the present study, weused SRA to examine if the coating length was critical forproducing a highly reproducible stroke model.

Materials and MethodsExperimental GroupsAnimal procedures were approved by the Institutional Animal Careand Use Committee of Shanghai Jiao Tong University, Shanghai,China. Twenty male Sprague-Dawley rats (SLAC, Shanghai, China)weighing 270 to 350 g were divided into 4 groups randomly(n�5/group). The sutures with different physical properties wereused in 4 groups to produce the MCAO model: Group 1, 4-0monofilament nylon suture without silicone coating; Group 2, 4-0suture with �2.0-mm silicone coating length; Group 3, 4-0 suturewith 2.0 to 3.3-mm silicone coating length; and Group 4, 4-0 suturewith �3.3-mm silicone coating.

Surgical ProcedureAnimals were anesthetized using 50/10 mg/kg ketamine/xylazine(Shanghai Pharmaceuticals Holding Co, Shanghai, China) intraperi-

Received August 17, 2011; accepted September 15, 2011.From the Departments of Radiology (Y.G., K.C.) and Neurology (H.L., G.-Y.Y.), Ruijin Hospital, School of Medicine, Shanghai, China; Med-X

Research Institute and School of Biomedical Engineering (Y.W., F.Y., H.L., G.-Y.Y.), Shanghai Jiao Tong University, Shanghai, China; ShanghaiInstitute of Applied Physics (Y.R., T.X.), Chinese Academy of Sciences, Shanghai, China; and the Buck Institute for Age Research (D.A.G., K.J.),Novato, CA.

Correspondence to Guo-Yuan Yang, MD, PhD, Med-X Research Institute, Shanghai Jiao Tong University, 1954 Hua-Shan Road, Shanghai 200030,China. E-mail gyyang0626@gmail.com

© 2011 American Heart Association, Inc.

Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.111.636456

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toneally. The middle cerebral artery was occluded as describedpreviously.9 A customized “T”-shaped PE-10 catheter was carefullyinserted into the internal carotid artery and fixed throughout thesubsequent imaging procedure.

Synchrotron Radiation ImagingThe experiment was conducted at beam line BL13W, ShanghaiSynchrotron Radiation Facility. The beam current was 145 mA andx-ray energy was 33.7 keV. A charged coupled device camera(13 �m/pixel) was used to obtain real-time angiographic imaging.Images were snapped at 30 frames/second. Animals were placed inthe right or left lateral decubitus position on a flexible platform. Atotal of 175 mg/mL nonionic iodine contrast agent (Omnipaque; GEHealthcare) was administered through a PE-10 catheter at aninjection rate of 100 �L/s. The total injection volume was �1 mLper rat.

Infarct Volume AnalysisRats were euthanized 24 hours after MCAO. Brains were isolatedand cut into 2-mm-thick coronal slices and stained with 2% wt/vol2,3,5-triphenyltetrazolium chloride (Sigma, St Louis, MO). Infarc-tion volume was measured using National Institutes of Health ImageJ software (Bethesda, MD) and calculated as described.9

StatisticsData were presented as means�SD. Statistical differences amonggroups were determined by 1-way analysis of variance withrepeated measures. A value of P�0.05 was considered to bestatistically significant.

Results

SRA of the Rat BrainThe entire vasculature of the left hemisphere includingpterygopalatine artery, internal carotid artery, middle cerebralartery (MCA), anterior choroidal artery, posterior cerebralartery, and small (13 �m diameter) arteries and small veinswas detected. The distance between the proximal posteriorcerebral artery/MCA, anterior choroidal artery/MCA, andMCA/anterior cerebral artery is 2.23�0.25 mm,1.50�0.18 mm, and 1.58�0.35 mm, respectively (n�14).The distance between branches of the MCA in the bibranchedMCA is 1.47�0.05 mm (n�6).

Figure 1. Synchrotron radiation angiography images were taken immediately after the injection of contrast agent. Images shown (top tobottom) were taken at 50 ms, 125 ms, 200 ms, and 275 ms. Normal cerebral vasculature (left); unsuccessful middle cerebral arteryocclusion (MCAO) using 4-0 suture without coating (center) and successful MCAO using silicone-coated 4-0 suture (right) are detected.

Figure 2. Synchrotron radiation angiography images how theextent of middle cerebral artery (MCA) occlusion using silicone-coated sutures with different coating lengths. The suture can bevisualized in these images as a thin white line in the internalcarotid artery. The MCA was only partially occluded when thesuture head was not coated (A) or the coating length was�2 mm (B). The MCA was successful occluded when the coat-ing length was 2.0 to 3.3 mm without affecting the hypothalamicartery (HTA), posterior cerebral artery (PCA), and anterior choroi-dal artery (AChA; C). The MCA was occluded when the coatinglength �3.3 mm together with AChA/PCA, and HTA (D).

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Effect of Suture Properties on MCAOWe first tested the effect of 4-0 nylon suture with or withoutsilicone coating. We found that contrast enhancement wasstill detected in the MCA when an uncoated suture was used(Figure 1). Next, we examined whether the length of siliconecoating would influence its ability to block the backflow fromcommunicating arteries. We demonstrated that the MCAcannot be occluded when the suture tip was not coated; whenthe silicone coating length was �2 mm, the MCA waspartially blocked; when the silicone coating length was 2.0 to3.3 mm, the MCA was completely occluded; when thecoating length was �3.3 mm, arteries including the anteriorchoroidal artery/posterior cerebral artery and hypothalamicartery were occluded (Figure 2). Further study revealed thecorrelation between suture coating length and infarct size(Figure 3). When the coating length was 2 to 3.3 mm,ischemia affected both the cortex and caudate–putamen withvery small deviation. Additionally, this coating length wasalso optimal for survival (2–3.3 mm [100%] versus �3.3 mm[25%]; P�0.05).

DiscussionThe ability to monitor vessel occlusion in real time mayensure uniformity in the location and completion of occlu-sion. Recently developed techniques such as MR angiographyprovide in vivo methods for imaging the cerebral circulationin rodents.10 However, the resolution of MR angiography isrelatively low, which is likely to preclude its use for moni-toring of MCAO in rodents.11 SRA provides a useful tool tostudy changes in cerebral blood flow and vascular morphol-ogy in real time in the rodent brain.

Early intraluminal MCAO models used silicone–rubber-coated and flame-blunted monofilaments.2 However, vari-ability in infarct size led to modification of sutures withdifferent coating materials.12 Additionally, heat-bluntedmonofilaments were found to be associated with a variableinfarction volume. Methods for comparing the effects ofdifferent occlusion techniques have been lacking. Our resultsshow that in a certain range of weight (we used 270- to 350-grats), the length of suture coating is a critical factor ingenerating reproducible infarct after MCAO. However, sev-eral disadvantages need to be considered. The imagingwindow of SRA is small (4�40 mm). Additionally, the SRAdevice is not universally available. Nevertheless, SRA pro-vides a valuable technological adjunct to the study of exper-imental stroke.

AcknowledgmentsWe thank Honglan Xie, Guohao Du, Bohua Xie, Jun Huang, andYaohui Tang for their assistance.

Sources of FundingThis work was supported by 973 program 2010CB834306 (G.-Y.Y.,Y.W.), the Shanghai Science and Technology Commission Program09140902400 (G.-Y.Y., Y.W.), KC Wong Foundation (G.-Y.Y.),and Shanghai Leading Academic Discipline Project S30203 (K.C.).

DisclosuresNone.

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Figure 3. Illustration of coating length of the suture in relation to the cerebral vasculature. Photographs show 2,3,5-triphenyltetrazoliumchloride-stained (red) brain sections, in which infarct areas remain unstained (white). Panel at the bottom right shows infarct volume(mm3) in controls (Cont) and after attempted middle cerebral artery occlusion with uncoated suture (No) and suture coated with siliconeto a length of �2.0 mm, 2.0 to 3.3 mm, and �3.3 mm. Data are mean�SD, n�5/group.

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David A. Greenberg, Kunlin Jin and Guo-Yuan YangYongjing Guan, Yongting Wang, Falei Yuan, Haiyan Lu, Yuqi Ren, Tiqiao Xiao, Kemin Chen,

Synchrotron Radiation AngiographyEffect of Suture Properties on Stability of Middle Cerebral Artery Occlusion Evaluated by

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