our study was performed on the basis of the guidelines set both by the u.s .
national institute of health and the recommendations of the committee on animal research at our institution .
ten pigs ( n = 10 , 30 - 35 kg ) were subjected to 90 minutes of occlusion of the left anterior descending coronary artery , and this was followed by 90 minutes of reperfusion . after performing a left lateral thoracotomy along the fifth intercostal space , the left anterior descending coronary artery was isolated distal to the first diagonal branch , and a snare loop made with 4 - 0 silk was placed in a slender plastic tube .
occlusion or reperfusion of the left anterior descending coronary artery was produced by simply fastening or releasing the snare loop .
the hearts were rapidly excised and placed in a bath of cold ( 4 ) cardioplegic solution .
the hearts were stored in formalin solution for at least 18 hour to let the possible early ventricular geometry changes occur before mr imaging , as was previously suggested ( 9 , 14 ) .
the dt - mri were acquired from the ten excised hearts by using a philips 3 t achieva scanner ( philips medical system , best , netherlands ) and an eight - channel head coil . the formalin - fixed porcine heart specimens were suspended in a cylinder filled with formalin to avoid tissue - air susceptibility artifacts . after the scout images were acquired on the four - chamber and two - chamber views , the short axis images of the left ventricle were obtained at the midventricular level for dt - mri .
the sensitivity encoding ( sense)-based echo - planar imaging technique was applied to shorten the image acquisition time and to alleviate any image distortion caused by susceptibility artifacts from the single - shot echoplanar imaging sequences .
the sense factor was chosen to be 2.4 in this study as a trade off between image distortion due to the high echo - planar imaging factor and the sense artifacts caused by a high sense factor , as was previously suggested ( 14 ) . with a b - value of 800 s / mm ,
the diffusion tensor images were obtained , respectively , for 6 , 15 and 32 diffusion gradient directions at the same midventricular level of each specimen .
the imaging parameters were as follows : te = 55 ms , tr = 5000 ms , number of slices = 5 , slice thickness = 1.13 mm , slice gap = 0 mm and the number of excitations = 1 .
three - dimensional reconstruction of the myocardial fibers was done by using the commercially available pride software package ( philips medical systems , best , netherlands ) . for each of the short - axis slices ,
the numbers of tracked fibers , the fractional anisotropy ( fa ) and the length of the tracked fibers in the left ventricle were measured using the pride software by an experienced cardiac radiologist who has 12 years of experience with quantitative analysis .
the fiber distribution was investigated with the fa magnitude and directional thresholds set at 0.15 and 40 , respectively ( 14 ) .
the image quality of the fiber tractography was assessed by two radiologists for qualitative analysis .
they scored each image on a 3-point scale : 1 ( poor ) , 2 ( moderate ) and 3 ( good ) .
the definition of each rating score is as follows : ' poor ' was visualization of the myocardium with frequent discontinuity except for in the infarcted myocardium ; ' moderate ' was visualization of the myocardium with partial discontinuity except for in the infarcted myocardium ; ' excellent ' was visualization of myocardial fibers without or with minimal discontinuity except in the infarcted myocardium .
the average of the scores for each image was used for qualitative analysis . for statistical analysis among the three paired data sets ,
all quantitative and qualitative comparisons were analyzed by using friedmann 's test and the wilcoxon - signed rank test for post - hoc consideration .
spss version 12.0 ( spss , chicago , il ) was used for the statistical calculation .
our study was performed on the basis of the guidelines set both by the u.s .
national institute of health and the recommendations of the committee on animal research at our institution .
ten pigs ( n = 10 , 30 - 35 kg ) were subjected to 90 minutes of occlusion of the left anterior descending coronary artery , and this was followed by 90 minutes of reperfusion . after performing a left lateral thoracotomy along the fifth intercostal space , the left anterior descending coronary artery was isolated distal to the first diagonal branch , and a snare loop made with 4 - 0 silk was placed in a slender plastic tube .
occlusion or reperfusion of the left anterior descending coronary artery was produced by simply fastening or releasing the snare loop .
the hearts were rapidly excised and placed in a bath of cold ( 4 ) cardioplegic solution .
the hearts were stored in formalin solution for at least 18 hour to let the possible early ventricular geometry changes occur before mr imaging , as was previously suggested ( 9 , 14 ) .
the dt - mri were acquired from the ten excised hearts by using a philips 3 t achieva scanner ( philips medical system , best , netherlands ) and an eight - channel head coil . the formalin - fixed porcine heart specimens were suspended in a cylinder filled with formalin to avoid tissue - air susceptibility artifacts . after the scout images were acquired on the four - chamber and two - chamber views , the short axis images of the left ventricle were obtained at the midventricular level for dt - mri . the sensitivity encoding ( sense)-based echo - planar imaging technique was applied to shorten the image acquisition time and to alleviate any image distortion caused by susceptibility artifacts from the single - shot echoplanar imaging sequences .
the sense factor was chosen to be 2.4 in this study as a trade off between image distortion due to the high echo - planar imaging factor and the sense artifacts caused by a high sense factor , as was previously suggested ( 14 ) . with a b - value of 800 s / mm ,
the diffusion tensor images were obtained , respectively , for 6 , 15 and 32 diffusion gradient directions at the same midventricular level of each specimen .
the imaging parameters were as follows : te = 55 ms , tr = 5000 ms , number of slices = 5 , slice thickness = 1.13 mm , slice gap = 0 mm and the number of excitations = 1 .
three - dimensional reconstruction of the myocardial fibers was done by using the commercially available pride software package ( philips medical systems , best , netherlands ) . for each of the short - axis slices ,
the numbers of tracked fibers , the fractional anisotropy ( fa ) and the length of the tracked fibers in the left ventricle were measured using the pride software by an experienced cardiac radiologist who has 12 years of experience with quantitative analysis .
the fiber distribution was investigated with the fa magnitude and directional thresholds set at 0.15 and 40 , respectively ( 14 ) .
the image quality of the fiber tractography was assessed by two radiologists for qualitative analysis .
they scored each image on a 3-point scale : 1 ( poor ) , 2 ( moderate ) and 3 ( good ) .
the definition of each rating score is as follows : ' poor ' was visualization of the myocardium with frequent discontinuity except for in the infarcted myocardium ; ' moderate ' was visualization of the myocardium with partial discontinuity except for in the infarcted myocardium ; ' excellent ' was visualization of myocardial fibers without or with minimal discontinuity except in the infarcted myocardium .
the average of the scores for each image was used for qualitative analysis . for statistical analysis among the three paired data sets ,
all quantitative and qualitative comparisons were analyzed by using friedmann 's test and the wilcoxon - signed rank test for post - hoc consideration .
spss version 12.0 ( spss , chicago , il ) was used for the statistical calculation .
the results of the quantitative analysis are summarized in table 1 . by increasing the numbers of diffusion - sensitizing gradient directions from 6 to 15 and to 32 on dt - mri , the mean
fa and standard deviation were significantly reduced ( p < 0.01 ) . for the evaluation of fiber tracking ,
the number of tracked fibers was significantly increased ( p < 0.01 ) and the length of the tracked fibers was also increased ( p < 0.01 ) with the increased number of the diffusion - sensitizing gradient directions ( figs . 1 - 3 ) . in the qualitative analysis ,
the image quality of the fiber tractography was significantly increased according to the increased number of the diffusion - sensitizing gradient directions ( p < 0.01 ) ( table 2 ) .
our result showed that higher numbers of diffusion - sensitizing gradient directions can provide more detailed information about the myocardial fiber structure on in vitro dt - mri at 3 t . to the best our current knowledge
, no consensus exists for the optimal number of diffusion - sensitizing gradient directions for dt - mri .
jones ( 19 ) insisted that the mean diffusivity , fa and tensor orientation requires a dt - mri sampling scheme in which there are at least 30 diffusion - sensitizing gradient directions by using monte carlo simulations .
our study also suggested that one should use as many unique sampling orientations as time will allow for applications such as fiber tractography .
( 13 ) demonstrated that dt - mri in the cervical spinal cord with using 15 diffusion - sensitizing gradient directions showed significantly better quality than that using 6 directions .
yet dti using 32 directions did not show a significant improvement of quality over that using 15 directions .
they suggested that the increased numbers of diffusion - sensitizing gradient directions in vivo dt - mri require longer scan times and there may be a greater risk for motion artifacts .
this is a first report on using a higher number of diffusion - sensitizing gradient directions for cardiac dt - mri .
most studies have used low diffusion resolution with employing 6 diffusion - sensitizing gradient directions for cardiac dt - mri .
wu et al . ( 14 ) recently used dt - mri with medium diffusion resolution with employing 15 diffusion - sensitizing gradient directions for the evaluation of myocardial fiber pathways in canine heart samples fixed in formalin .
they revealed that the long fiber pathways were found to predominantly run circumferentially with small helix angles , and this dominated the fiber architecture in the myocardium .
however , they only investigated the myocardial fiber pathway distribution and they did not analyze the effect of medium diffusion resolution for the dt - mri quality for the evaluation of myocardial anisotropy and fiber tracking . in this study , we demonstrated the effect of high diffusion resolution by using 32 diffusion - sensitizing gradient directions for in vitro dt - mri .
we suggest that high resolution dt - mri may be used as a tool for delineating and monitoring the detailed myocardial fiber structure after a myocardial infarction has responded to various treatments such as stem cell therapy .
although the advantages of mri at 3 t over the 1.5 t systems have not been established for the use of cardiac dt - mri , a better depiction of fiber tracts at 3 t compared with 1.5 t has been demonstrated with performing high spatial resolution dt - mri in brain ( 20 - 22 ) . the sensitivity - encoded ( sense )
mr technique , as a parallel imaging method , is now feasible with using a commercial machine and with no dedicated hardware . using the sense technique ,
the duration of the echo train is reduced by faster filling of the k - space and so this results in a wider bandwidth in the phase - encoded direction ; therefore , the susceptibility artifacts are reduced ( 23 , 24 ) . to the best of our knowledge , there is no consensus concerning the optimum method for evaluating the quality of dt - mri .
( 25 ) investigated the optimum imaging parameters of dt - mri in brain , and they quantitatively evaluated the numbers of reconstructed fibers and the visual image quality . in a similar fashion , we evaluated the qualities of the dt - mri with using different numbers of diffusion - sensitizing gradient directions by quantifying the numbers and length of the tracked fibers , as well as by visually comparing the image quality of the fiber tractography .
our results demonstrated that the numbers and length of the tracked fibers were significantly increased with the increased number of diffusion - sensitizing gradient directions , which suggests that the image quality of the fiber tractography was significantly improved .
however , the validity of using fiber tracking as a means to determine the accuracy of dt - mri measurements was not proven experimentally . without loss of generality
, the dti - derived fa index ( 26 ) , which is a commonly used rotationally invariant index of diffusion anisotropy , is examined as a function of the left ventricular wall depth and the circumferential and longitudinal locations .
jiang et al . ( 27 ) reported that the diffusion anisotropy measured by dt - mri in the fixed sheep myocardium remained relatively constant from the epicardium to the midwall and then it decreased steadily toward the endocardium .
( 12 ) also demonstrated that in vivo dt - mri of the postinfarct myocardium in patients with myocardial infarction revealed a significant decrease in fa , indicating there was altered tissue integrity .
furthermore , the redistribution of the fiber architecture correlated with the infarct size and the left ventricular function .
therefore , exactly measuring the fa is important for characterizing such tissue parameters as the extracellular volume fraction as well as the fiber orientation on cardiac dt - mri .
chang et al . ( 28 ) revealed , by using the histogram method , that the image quality of the fa map of a brain was significantly improved as the number of diffusion - sensitizing gradient directions increased .
they also reported that that the histogram showed that the fa values and their variance were increased as the number of diffusion gradient directions decreased , and this was probably due to the overestimation of the fa values .
it was also shown in our study that the mean fa and its standard deviation were significantly reduced with the increased number of diffusion - sensitizing gradient directions .
first , we have no direct histopathological correlations to validate our findings . however , it is difficult to precisely quantify the numbers and the length of myocardial fibers on histopathological exam .
the increased scan time may have produced more motion - related artifacts on the in vivo dt - mri .
optimizing the number of diffusion - sensitizing gradient directions should be considered as a tradeoff between increasing the image quality and minimizing the scan time for in vivo dt - mri . advanced mr technology
will be required to overcome such problems as the low signal - to - noise ratio and the long scan time for in vivo dt - mri .
third , we did not consider several important parameters such as the b - value , the numbers of excitations and the slice thickness for cardiac dt - mri .
further study will be needed to combine several individual parameters for optimizing the acquisition of in vivo dt - mri before it can be applied to clinical settings .
finally , we did not separately evaluate the alterations in tissue integrity , such as the fa of both the infarcted and normal myocardium , because the purpose of our study was only to evaluate the effect of the number of diffusion - sensitizing gradient directions on the image quality of dt - mri . from this study , we conclude that the image quality of in vitro dt - mri is significantly improved as the number of diffusion - sensitizing gradient directions is increased .
 objectivewe wanted to evaluate the effect of the number of diffusion - sensitizing gradient directions on the image quality for evaluating myocardial anisotropy and fiber tracking by using in vitro diffusion tensor mr imaging ( dt - mri).materials and methodsthe dt - mr images , using a sense - based echoplanar imaging technique , were acquired from ten excised porcine hearts by using a 3 t mr scanner . with a b - value of 800 s / mm2 , the diffusion tensor images were obtained for 6 , 15 and 32 diffusion - sensitizing gradient directions at the midventricular level . the number of tracked fibers , the fractional anisotropy ( fa ) , and the length of the tracked fibers were measured for the quantitative analysis . 
 two radiologists assessed the image quality of the fiber tractography for the qualitative analysis.resultsby increasing the number of diffusion - sensitizing gradient directions from 6 to 15 , and then to 32 , the fa and standard deviation were significantly reduced ( p < 0.01 ) , and the number of tracked fibers and the length of the tracked fibers were significantly increased ( p < 0.01 ) . the image quality of the fiber tractography was significantly increased with the increased number of diffusion - sensitizing gradient directions ( p < 0.01).conclusionthe image quality of in vitro dt - mri is significantly improved as the number of diffusion - sensitizing gradient directions is increased . 
MATERIALS AND METHODS
Animal Preparation
MR Imaging
Data Analysis
RESULTS
DISCUSSION