Optical measurement of mouse strain differences in cerebral blood flow using indocyanine green

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1 Journal of Cerebral Blood Flow & Meabolism (15), ISCBFM All righs reserved X/15 $. BRIEF COMMUNICATION Opical measuremen of mouse srain differences in cerebral blood flow using indocyanine green Hye-Min Kang 1, Inkyung Sohn 1, Seunggyu Kim, Daehwan Kim, Junyang Jung 1, Joo-Won Jeong 1 and Chan Park 1 mice have more cerebral arerial branches and collaerals han mice. We measured and compared blood flow dynamics of he middle cerebral arery () in hese wo srains, using noninvasive opical imaging wih indocyanine green (ICG). Relaive maximum fluorescence inensiy (I max ) and he ime needed for ICG o reach I max in he of C57BL/c were lower han ha in mice. Moreover, he mean ransi ime was significanly lower in han in mice. These daa sugges ha he higher number of arerial branches and collaerals in mice yields a lower blood flow per cerebral arery. Journal of Cerebral Blood Flow & Meabolism advance online publicaion, 1 April 15; doi:1.18/jcbfm.15.5 Keywords: cerebral blood flow measuremen; cerebral hemodynamics; imaging; middle cerebral arery; opical imaging INTRODUCTION mice have more severe issue damage, and ake longer o recover from sroke han mice. 1, These differences have been aribued o dispariies in branch densiy and collaeral inerconnecions of he cerebral vasculaure. Anaomically, mice have more collaeral anasomoses han oher mouse srains; however, here is lile physiological daa showing differences in cerebral blood flow (CBF) dynamics beween and mice. Measuremen of CBF is imporan for he diagnosis and monioring of vascular diseases. The fluorescen dye indocyanine green (ICG) can be used o measure CBF, and can significanly enhance he signal-o-noise raio. Moreover, he kineics of he ICG bolus can improve he deecion of deep organs and issue blood flow in experimenal animals 5,6 and humans. 7,8 A recen sudy showed ha near infrared imaging of ICG fluorescence, combined wih a ime-series analysis of is molecular dynamics, can be useful for CBF analysis. 9 Thus, in he curren sudy, we visualized blood flow in he cerebral areries and venous sinuses using ICG-opical imaging, and measured several feaures of blood flow dynamics o assess CBF differences in cerebral areries of and mice. To he bes of our knowledge, his is he firs sudy showing differences in CBF in wo mouse srains. MATERIALS AND METHODS Animal Preparaion Eigh-week-old male and mice were obained from Daehan Biolink (Gyeonggi-do, Republic of Korea). In hese wo mice srains, males have he same blood pressure. 1 Animals were anesheized wih keamine (.1 mg/g w) and xylazine (.1 mg/g w). We measured sysemic physiologic parameers of blood relevan for resing CBF (Supplemenary Informaion S1). Body emperaures were moniored by a recal probe, and mainained a 7 C using a emperaure-conrolled heaing pad (Harvard Apparaus, Hollison, MA, USA). All procedures were approved by he Insiuional Animal Care and Use Commiee of he Kyung Hee Universiy. We performed all experimenal procedures and handled experimenal animals according o he ARRIVE guidelines (hp:// 11 Indocyanine Green Imaging Under aneshesia, an incision was made o expose he enire skull. Then, mice were placed under an 8-nm band-pass filer CCD camera. A soluion ( μl) of ICG (.1 mg/g w) was injeced manually ino he ail vein (he rae of injecion: 8 μl/s) using a 1-gauge needle, and he head was illuminaed wih 76 nm wavelengh lighs. Time series of ICG fluorescence signal was acquired every 16 ms for minues. Using a cusomized sofware (Vieworks Co., Ld., Anyang, Republic of Korea), he iniial 1 frames were used o generae blood flow maps as previously repored by Ku e al. 9 The T rising value was calculaed as he ime beween T arrival (ime of firs appearance of ICG fluorescence, i.e., arrival of he bolus) and T max (ime of maximum inensiy). The slope of he firs peak in he ime-inensiy curve was used o calculae he blood flow index (BFI), dividing he relaive fluorescence inensiy a T peak (he firs peak ime) by T rising. Mean ransi ime (MTT) was calculaed as he cener of graviy of he dynamic curve. 1 We used he signals during he 16 seconds period afer T arrival for he calculaion. 1 Deparmen of Anaomy and Neurobiology, Biomedical Science Insiue, School of Medicine, Kyung Hee Universiy, Seoul, Korea and R&D Cener, Vieworks Co., Ld., Anyang, Korea. Correspondence: Dr C Park, Deparmen of Anaomy and Neurobiology, Biomedical Science Insiue, School of Medicine, Kyung Hee Universiy, #1 Hoeki-dong, Dongdaemun-gu, Seoul 1-71, Korea. psychan@khu.ac.kr This work was suppored by he Basic Science Research Program of he Naional Research Foundaion of Korea (NRF) funded by he Minisry of Educaion, Science and Technology (NRF-1R1A1A6667). Received 1 Ocober 1; revised 17 February 15; acceped March 15

2 Afer adjusing he baseline inensiy o zero, he following equaion was applied: P UIðÞ MTT ¼ P I ðþ Differences in CBF in wo mouse srains H-M Kang e al Saisical Analysis Daa are given as he mean ± s.e.m. for n number of mice ((n =6) per srain) and were analyzed using Suden s -ess. RESULTS Srucural Differences in he Arerial Tree beween and Mice Two minues afer inravenous ICG injecion, raw images were acquired and sreamed o a compuer. In he raw fluorescen image, blood vessels appeared as brigh areas. We made a represenaive movie o show he kineics of blood flow (Supplemenary Informaion S). From he serial images, we creaed a filered ime-series sack image, and noiced ha he number of disal arerial branches appeared o differ beween and mice during angiographic analyses (Figure 1A). Therefore, we measured he number of branching poins from he middle cerebral arery () and anerior cerebral arery. Afer magnifying he image on he monior, we delineaed he arerial rees on he overlying layer. The vessels delineaed in his image were analyzed using ImageJ and he Skeleon plugins (hp://rsbweb.nih.gov/ij/). Our daa showed ha mice had significanly more branching poins (.5 ±.195) han mice (1 ± 1.65) (Po.5) (Figure 1B). See more deails in Supplemenary Informaion S. Analysis of Single-Pixel Indocyanine Green Dynamics in Cerebral Vessels and Comparison Beween Srains We seleced one pixel in he firs branching area of he, and one in he superior sagial sinus () (Figure 1C), in fluorescence images. Then, we examined he dynamics of each pixel (Figure 1D), which ypically consised of an iniial peak followed by subsequen peaks produced by sysemic recirculaion (Figure 1E). Aferwards, we exraced a T rising value from he firs peak as a represenaive parameer of he saus of issue blood supply. T rising was calculaed by subracing T arrival from T max (Figure 1E). To idenify differences in T max of each pixel, we reconfigured he ICG dynamics graph (Figure 1F) o show ha each poin had a differen T max. We nex normalized each dynamic value, and compared he T max beween. and., and beween. and.. Using his approach, we deermined ha he had a larger difference in T max han he (Figures 1G and 1H). Thus, analysis of singlepixel dynamics showed a probable difference in CBF in he and beween he wo mice srains. Blood Flow Feaure Maps and Comparison Beween Srains To analyze saisical differences in CBF beween and mice, we assessed blood flow feaures in he following regions of ineres (ROIs;.5 mm diameer): he firs branching area (yellow circle) appearing on he laeral area of he parieal bones in boh hemispheres, and wo ROIs in he (blue circles) around he juncion wih he cerebral veins (Figure A). Values for each blood flow feaure (T rising, I max, BFI, and MTT) were mapped ono he above-menioned vascular brain regions (Figure B). These values were hen calculaed and compared in he and (Figure C). In T rising maps, he overall T rising value of he in mice (.6 ±.118) was larger han in mice (.16 ±.57) (Po.1). This suggess ha CBF akes longer o reach I max in han in mice. In addiion, he I max value of he in mice (5,7.6 ±.98) was higher han in mice (,5.9 ± ) (Po.1), suggesing ha more blood flows in he s of han in mice. The BFI was calculaed by deermining he change in inensiy of he maximum value/t rising. 9 Ineresingly, mice showed a endency for slighly higher BFI, alhough BFI was no significanly differen beween he wo srains (Figure C). The MTT was defined as he ransiion ime afer he bolus arrives a a region. 9,1 The MTT for he in mice (7.7 ±.8) was larger han in mice (7.16 ±.1) (Po.5), suggesing ha a bolus of ICG in he of he srain akes longer ime o ransi hrough he arery. To evaluae CBF in he disal branches of he and microvessels in corical issue, we delineaed mm diameer ROIs covering hese areas on boh hemispheres. The ROI ceners were posiioned mm poserior and mm laeral from bregma (Supplemenary Informaion S). Ineresingly, T rising and I max were significanly higher in he mice; however, MTT and BFI were no significanly differen beween srains. Similar corical values for BFI and MTT sugges ha blood flow rae in he microvessels of he corex is similar beween he wo srains. DISCUSSION Alhough previous sudy repored ha blood flow assessed by [ 1 C]iodoanipyrine auoradiography in he laeral corex (he erriory) was no significanly differen beween and, 1 we hypohesized ha differences in branch densiy and collaerals could lead o dissimilariies in CBF across mouse srains. To examine his hypohesis, we adoped a very sensiive opical imaging mehod using he near-infrared fluorescence dye ICG, combined wih a ime-series analysis of ICG molecular dynamics. There were differences in ICG dynamics beween srains, which were successfully deeced using four dynamic parameers (i.e., T rising, I max, MTT, and BFI). To our knowledge, his is he firs sudy examining differences in CBF beween and mice. Our sudy revealed ha cerebral arerial branching and collaerals deermined he values of he various CBF parameers. Thus, since cerebral arerial branching and collaerals differ across mouse srains, CBF parameers differ according o heir geneic backgrounds. Single-pixel ICG dynamics in he vessels of he wo srains revealed ha differences in CBF could be analyzed saisically. Paricularly, i was deermined ha T rising and I max were higher in he of han in mice. However, he BFI, or he slope of he firs peak, did no differ significanly beween srains. Consequenly, he infusion rae of blood in he was no significanly differen beween and mice. However, he elevaed I max in he of mice suggess ha hese mice have a larger volume of blood han mice in a branch of he. A possible explanaion is ha, during developmen, he number of second- and hird-order branches is lower in, while he diameer of second-order branches is larger, hus more blood may flow in a branch of he in mice. In addiion, our daa show ha he MTT in he of mice was higher han in mice. This could be explained by he fac ha a higher volume of blood akes longer ime o ransi hrough an arery, and he srain has more arerial branches and collaerals, which may imply shorer ransi imes for blood in he. In our sudy, mice reached faser an ICG peak in he, bu he rae did no differ in he. Thus, we compared CBF of he disal par of he and corical microvessels in he cener area of he hemisphere. The resuls showed ha here were no differences in MTT beween he wo srains, suggesing ha Journal of Cerebral Blood Flow & Meabolism (15), ISCBFM

3 Differences in CBF in wo mouse srains H-M Kang e al Fluorescence inensiy (a.u.) Fluorescence inensiy (a.u.) T rising Imax T arrival T max Fluorescence inensiy (a.u.) Normalized fluorescence inensiy (a.u.) Normalized fluorescence inensiy (a.u.) Figure 1. Time-series sack image and single-pixel dynamics of wo mouse srains. (A) Represenaive filered ime-series sack image of he cerebral areries. There are fewer visible cerebral arery branches in. (B) The number of cerebral arery branching poins was significanly differen beween he wo srains. Daa represen he mean ± s.e.m. (Suden s -es, Po.1). (C) Time-series image afer ail vein injecion of indocyanine green (ICG). Seleced pixels are indicaed by colored circles (pink and sky blue: firs branching poin of he middle cerebral arery (); red and blue: superior sagial sinus (). (D) ICG dynamics of each pixel afer he injecion of an ICG bolus. (E) Inerpolaed dynamics were ploed, and T arrival and T max (for he calculaion of T rising ) are indicaed in he ime axis. I max is indicaed on he fluorescence inensiy axis. (F) Expanded graph showing he ime window of ineres ( o 5 seconds) of (D). (G) Normalized graph of ICG dynamics a he firs branching poin showing T max differences beween he wo srains (double-headed arrow). (H) Normalized graph of ICG dynamics a he showing T max differences beween he wo srains (wo arrows). microvessel neworks in he cerebral corex have similar blood flow raes. Ineresingly, mice showed a endency for slighly higher corical microvessel area densiy, 1 which may explain he similar peak ime of ICG s for and. Several anaomic sudies have showed ha differences in cerebral vasculaure (e.g., branching and collaerals) in mouse srains can influence infarc size, and he recovery ime afer soke. 1, In he curren sudy, we showed ha wo mouse srains 15 ISCBFM Journal of Cerebral Blood Flow & Meabolism (15), 1 5

4 Differences in CBF in wo mouse srains H-M Kang e al Arery only Whole vessels Arery only Whole vessels T rising (s) 1 MTT BFI Imax T rising I max (a.u.) BFI (a.u.) MTT (s) Figure. Types of cerebral blood flow (CBF) maps, and comparison of CBF parameers in and mice. (A) Seleced regions of ineres (ROIs) are indicaed by colored circles (yellow: firs branching area of middle cerebral areries (s) in boh hemispheres; sky blue: wo randomly seleced areas of he superior sagial sinus ()). (B) Represenaive CBF maps of he and he. The firs branching area of he and he were seleced from he high inensiy area of he regions, and were processed using he image analysis program o creae CBF maps of he seleced area. Seleced area maps were rearranged on he ime-series sack image o indicae he locaion of each area in he and he. In each map, seleced ROIs are indicaed as yellow and blue circles, in he and he, respecively. (C) The averages of he blood flow parameers for each ROI were compared beween mouse srains. T rising, I max, and mean ransi ime (MTT) parameers showed significan differences in he beween he wo mouse srains. Daa represen he mean ± s.e.m. (Suden's -es, Po.5; Po.1; Po.1). BFI, blood flow index. have differen physiologic paerns of arerial blood flow, using ICG opical imaging. Our daa showed ha a larger volume of blood flows in a branch of he in, which explains why a blockage of a branch of cerebral arery in could induce a bigger infarcion han. Oher opical mehods o measure CBF include Laser Doppler flowmery, and Laser Speckle imaging. These wo echniques measure he velociy of flow of red blood cells. 15 Laser Doppler flowmery lacks on sensiiviy o esimae regional CBF, and only deecs relaive changes, 9 while Laser Speckle imaging is usually Journal of Cerebral Blood Flow & Meabolism (15), ISCBFM

5 used o visualize he vasculaure and make an image of relaive blood flow. 15 Indocyanine green provides accurae and reliable idenificaion of feeder and drainer vessels. This informaion is of grea use o idenify arery and vein. In addiion, ICG imaging provides informaion of blood volume. 15 Indocyanine green image shows a much sronger signal in he larger vessels compared o small vessels wih same blood flow rae. In his sudy, we used he properies of ICG o delineae he areries and veins, and measured CBF parameers on a specific region of he brain. In fuure sudies, we will use his noninvasive opical imaging mehod o examine he effec of neurovascular-relaed diseases on he perfusion rae of vessels in specific brain region. AUTHOR CONTRIBUTIONS HK and CP conceived and designed he experimens; HK and IS performed he experimens; HK, SK, DK, JJ, JWJ, and CP analyzed he daa; HK and CP wroe he paper; all auhors reviewed he manuscrip before submission. DISCLOSURE/CONFLICT OF INTEREST The auhors declare no conflic of ineres. ACKNOWLEDGMENTS The auhors hank Dr. Yujung Kang (R&D cener, Vieworks Co., Ld.) for reviewing he manuscrip and inerpreaion of daa. REFERENCES 1 Majid A, He YY, Gidday JM, Kaplan SS, Gonzales ER, Park TS e al. Differences in vulnerabiliy o permanen focal cerebral ischemia among common mouse srains. Sroke ; 1: Zhang H, Prabhakar P, Sealock R, Faber JE. Wide geneic variaion in he naive pial collaeral circulaion is a major deerminan of variaion in severiy of sroke. J Cereb Blood Flow Meab 1; : 9 9. Differences in CBF in wo mouse srains H-M Kang e al Chalohorn D, Faber JE. Formaion and mauraion of he naive cerebral collaeral circulaion. J Mol Cell Cardiol 1; 9: Chalohorn D, Clayon JA, Zhang H, Pomp D, Faber JE. Collaeral densiy, remodeling, and VEGF-A expression differ widely beween mouse srains. Physiol Genomics 7; : Kuebler WM, Sckell A, Habler O, Kleen M, Kuhnle GE, Wele M e al. Noninvasive measuremen of regional cerebral blood flow by near-infrared specroscopy and indocyanine green. J Cereb Blood Flow Meab 1998; 18: Kang Y, Choi M, Lee J, Koh GY, Kwon K, Choi C. Quaniaive analysis of peripheral issue perfusion using spaioemporal molecular dynamics. PLoS One 9; : e75. 7 Keller E, Nadler A, Alkadhi H, Kollias SS, Yonekawa Y, Niederer P. Noninvasive measuremen of regional cerebral blood flow and regional cerebral blood volume by near-infrared specroscopy and indocyanine green dye diluion. Neuroimage ; : Kang Y, Lee J, Kwon K, Choi C. Dynamic fluorescence imaging of indocyanine green for reliable and sensiive diagnosis of peripheral vascular insufficiency. Microvasc Res 1; 8: Ku T, Choi C. Noninvasive opical measuremen of cerebral blood flow in mice using molecular dynamics analysis of indocyanine green. PLoS One 1; 7: e88. 1 Mouse Phenome Daabase a The Jackson Laboraory. hp://phenome.jax.org/ db/qp?rn=views/measplo&brieflook = 1511&projhin = Jaxwes1. 11 Kilkenny C, Browne WJ, Cuhill IC, Emerson M, Alman DG. Improving bioscience research reporing: he ARRIVE guidelines for reporing animal research. PLoS Biol 1; 8: e11. 1 Gobbel GT, Cann CE, Fike JR. Measuremen of regional cerebral blood flow using ulrafas compued omography. Theoreical aspecs. Sroke 1991; : Lieber A, Wabniz H, Seinbrink J, Moller M, Macdonald R, Rinneberg H e al. Bedside assessmen of cerebral perfusion in sroke paiens based on opical monioring of a dye bolus by ime-resolved diffuse reflecance. Neuroimage 5; : Ward NL, Moore E, Noon K, Spassil N, Keenan E, Ivanco TL e al. Cerebral angiogenic facors, angiogenesis, and physiological response o chronic hypoxia differ among four commonly used mouse srains. J Appl Physiol 7; 1: Towle EL, Richards LM, Kazmi SM, Fox DJ, Dunn AK. Comparison of indocyanine green angiography and laser speckle conras imaging for he assessmen of vasculaure perfusion. Neurosurgery 1; 71: Supplemenary Informaion accompanies he paper on he Journal of Cerebral Blood Flow & Meabolism websie (hp:// com/jcbfm) 15 ISCBFM Journal of Cerebral Blood Flow & Meabolism (15), 1 5