eight 23-year - old male beagles ( mean body weight ; 10.51  1.48 kg , range ; 9.711.5 kg ) 
 were used . all dogs were clinically healthy based on a physical examination , complete blood 
 counts , biochemistry , urinalysis , thoracic and abdominal radiography , and abdominal 
 ultrasonography . there were unremarkable findings on lateral and cranio - caudal radiographs 
 of thoracic and pelvic limbs . the dogs were housed individually and were provided commercial 
 food and water ad libitum . after a 24 hr fast , the dogs were given intramuscular injections of a combination of 1.5 
 mg / kg zolazepam / tiletamine ( zoletil , virbac , carros , france ) and 0.03 mg / kg medetomidine 
 ( domitor , orion corp . conventional b - mode ultrasonography and ceus were conducted in left humerus , radius , femur 
 and tibia using the same 10 mhz linear probe and ultrasound equipment ( prosound alpha 7 , 
 aloka , tokyo , japan ) . the dog was positioned in a right lateral recumbency , and each 
 examining limb was extended toward the operator and fastened . a conventional b - mode 
 examination was performed to localize the scanning region for ceus . the transducer was 
 placed over the lateral plane from the shoulder joint to the humeral diaphysis proximally 
 covered by the triceps muscle ; the lateral plane from the proximal radial metaphysis to the 
 diaphysis proximally covered by the extensor digitorum communis muscle and distally by the 
 abductor pollicis longus muscle ; the lateral plane from the stifle joint to the femoral 
 diaphysis proximally covered by the tensor fasciae latae muscle and distally by the vastus 
 lateralis muscle ; or the lateral plane from the stifle joint to tibial diaphysis proximally 
 covered by the tibialis cranialis , respectively . the longitudinal image of each bone surface 
 including muscle was displayed on a monitor , with positioning so that the proximal 
 metaphysis was on the right and the diaphysis was on the left . second higher harmonics were performed in 
 the extended pure harmonic detection mode for ceus . the transmitted energy was reduced to a 
 magnitude of 7% with a 0.07 mechanical index . intrusion depth was set to 2.03.5 cm to 
 visualize the muscle according to the appendicular regions . the pulse repetition frequency 
 was set to 15 hz with a 61% gain value . contrast medium ( sonovue , bracco , milan , italy ) was 
 administered by bolus injection into the cephalic vein via a three way stopcock and a 
 20 gauge intravenous catheter . a 0.5 ml ( 2.5 mg ) or 1 
 ml ( 5 mg ) of contrast medium was injected through a 1 ml 
 syringe into each dog , immediately followed by a bolus injection of 5 ml 
 saline . at the same time , dynamic sequences were stored for up to 110 sec by 
 keeping the transducer in the same location . ceus was completed in all dogs within 2 hr 
 after opening the contrast medium bottle , and the contrast medium was agitated before 
 injection into other dogs . a single appendicular region was examined in each dog on the same 
 day , and the other regions were examined at 2-day intervals . all ceus was performed by one 
 examiner ( ojy ) , and quantitative and qualitative evaluations of all ceus were performed 
 three times by two reviewers ( ojy , jsh ) blinded to the scanned region . all dynamic cine 
 loops were evaluated using the installed software ( sop - alpha7 - 14 , aloka ) , which displayed 
 the acoustic intensity according to time . region of interest ( roi ) in an arc shape of 1.1  
 2.9 mm was manually defined over the muscle to acquire the tic . quantitative corresponding parameters of the tic , 
 including pi , auc and tp , were obtained from roi . tp was defined as the duration from the start of contrast agent injection to pi . auc was calculated as the area of the tic using integration . the protocol used in this study was approved by the animal care and use committee of 
 chonnam national university , and the animals were cared for in accordance with the 
 guidelines for animal experiments of chonnam national university . a statistical analysis was performed using the spss statistical program ( spss statistics 
 version 21 , ibm corp . analysis of reproducibility between reviewers 
 was performed using the intra - class correlation coefficient test ( icc ) . icc values were 
 evaluated by the criteria : poor reliability , < 0.4 ; fair to good , 0.40.75 ; and excellent , 
 > 0.75 . repeated - measures analysis of variance 
 was used to investigate the intra- and inter- reproducibility coefficients of the tic 
 parameters including pi , tp and auc and inter- reproducibility coefficients of vascularity 
 score among four appendicular regions in normal dogs . the mean blood pressure in eight dogs was 145.5  7.52 mmhg before anesthesia and 126.38  
 5.90 mmhg after anesthesia . transient increase of vascular intensity and then disappearance 
 was easily observed from all skeletal muscles after injecting the contrast medium ( fig . 1.contrast enhanced ultrasonography on harmonic detection mode before ( a ) and after ( b ) 
 contrast agent injection at the skeletal muscle of the humerus . transient increase of 
 vascular intensity ( arrows ) and then disappearance was easily observed from all 
 skeletal muscles after injecting the contrast medium . ) . contrast enhanced ultrasonography on harmonic detection mode before ( a ) and after ( b ) 
 contrast agent injection at the skeletal muscle of the humerus . transient increase of 
 vascular intensity ( arrows ) and then disappearance was easily observed from all 
 skeletal muscles after injecting the contrast medium . a total of 1,711 frames of ceus were obtained from roi over 110 sec , and a typical 
 parabolic shape to the tic was generated from muscle area ( fig . 2.conventional ultrasonography on color doppler mode ( a ) and contrast enhanced 
 ultrasonography on harmonic detection mode ( b ) of radius . region of interest placed 
 over the muscle on contrast enhanced ultrasonography and timeintensity curves ( c ) . the muscle area had a typical parabolic curve , which consisted of inflow , outflow and 
 peak intensity . ) . the mean values of pi , auc and tp measured from muscle were not significantly 
 different among the four appendicular regions ( table 
 1table 1.mean values of time - intensity curve parameters in appendicular muscles after 
 injection of 0.5 ml or 1 ml contrast agentpi ( level)auctp ( msec)0.5 ml1 ml0.5 ml1 ml0.5 ml1 mlhumerus28.84  9.6922.62  4.201,642.39  643.231,474.06  372.8040,763  10,27438,576  10,983radius19.86  8.9216.15  4.441,402.64  851.85838.91  366.6737,649 5,97643,101  11,463femur23.25  14.3628.31  10.371,786.42  1,279.552,041.50  769.3643,969  5,97638,576  8,281tibia16.40  6.1818.50  1.601,143.29  475.521,309.10  280.7642,890  10,37345,770 no significant difference in mean peak intensities , areas under the curve or 
 tp was observed within each skeletal muscle of the four regions , according to the dosage of 
 contrast medium . in quantitative analysis , the mean intra- and inter - reproducibility 
 coefficients of pi , auc and tp are represented in table 
 2table 2.mean intra- and inter - reproducibility coefficients of peak intensity , area under 
 the curve and time to peak intensity of the soft tissue in each appendicular bone 
 region using 0.5 ml or 1 ml contrast agent in 
 appendicular muscles0.5 ml1 mlintra - reproducibility coefficientsinter - reproducibility coefficientsintra - reproducibility coefficientsinter - reproducibility coefficientspitpaucpitpaucpitpaucpitpauchumerus0.7590.8270.8350.8710.8640.8060.8590.9720.9350.8710.9840.906radius0.9060.8080.8320.7550.9520.8600.9060.9980.9420.7650.9620.860femur0.9240.9700.9310.9330.6780.9270.9241.0000.9710.860.6780.927tibia0.7420.7440.8010.8990.7510.8560.7720.7740.8410.950.8910.885 . conventional ultrasonography on color doppler mode ( a ) and contrast enhanced 
 ultrasonography on harmonic detection mode ( b ) of radius . region of interest placed 
 over the muscle on contrast enhanced ultrasonography and timeintensity curves ( c ) . the muscle area had a typical parabolic curve , which consisted of inflow , outflow and 
 peak intensity . assessing blood perfusion is meaningful to predict prognosis or monitor progression of 
 fracture healing . the imaging modality has to be sensitive enough to detect low perfusion 
 for an accurate assessment of blood flow in muscle , because normal skeletal muscle perfusion 
 is very low at rest . the ultrasound contrast 
 medium , in principle , stays strictly intravascularly , and each single microbubble of 
 contrast medium can be detected using ceus , independent of velocity and volume of blood flow 
 . thus , ceus has the capacity to assess skeletal muscle perfusion accurately 
 not only at rest under normal conditions but also in fracture healing . a second - generation 
 contrast agent was selected in this study , because this agent coated with inert gas is more 
 stable allowing longer persistence in the blood stream than first - generation agents coated 
 with air . the size of sulfur hexafluoride - filled microbubbles is uniform in this contrast 
 medium , and this agent has prolonged and excellent stability with a vial time of 6 hr and a 
 half time of 6 min in peripheral blood . in 
 contrast to the first - generation contrast agents which become disrupted and release a large 
 amount of acoustic emission at a high mechanical index , low mechanical index imaging using 
 second - generation agents minimizes the microbubble burst to enhance the contrast effect and 
 produces a nonlinear oscillation which provides for harmonic frequencies for a longer 
 duration . these non - destructive contrast agents 
 allow for continuous real - time imaging and quantitative evaluation of blood perfusion by tic 
 analysis . the mean values of pi and auc represent the blood volume , and tp determines blood velocity 
 . pi and auc in skeletal muscle tended to be 
 higher in the humerus and femur than those in the radius and tibia , but no significant 
 difference was observed . tp in the thoracic limbs tended to be more rapid ( about 4 min ) than 
 that in the pelvic limbs ; however , no significant difference among the 4 regions was 
 observed . thus , blood volume and velocity in skeletal muscle were not significantly 
 different in the 4 appendicular regions . although a statistical analysis was not performed in this study , 
 ceus seemed to be more sensitive than color doppler for detecting small vessels and low 
 blood velocities , because ceus showed distinct vessel signals , not presented by color 
 doppler image in skeletal muscle . in addition , there are no doppler - specific artifacts , such 
 as aliasing and blooming in ceus . severe soft tissue trauma and circulatory disturbances are 
 one of the common causes of impediment to successful bone healing , because the fracture site 
 is initially supplied blood by extraosseous angiogenesis . in addition , excessive damage to 
 nutrient and periosteal vessels passing through cortical bone lead to delayed union .therefore , quantitative tic analysis of skeletal 
 muscular vascularization could provide early prognostic information about fracture 
 healing . a standard protocol 
 for using contrast agent in skeletal muscle is not established , even in humans , and many 
 different doses , such as 2.4 ml , 4.8 ml and 5 
 ml , with diverse injection methods including bolus injection and 
 continuous rate infusion have been applied [ 1 , 2 , 9 , 11 , 16,17,18 , 20 ] . in the present study , there was no significant 
 difference in pi , auc or tp between the two doses . consequently , 0.5 ml of 
 contrast medium may be more practical for assessing skeletal muscle perfusion . furthermore , quantitative assessment of skeletal muscle perfusion using tic values had 
 accuracy and objectivity with high reproducibility . first , this study was performed under general anesthesia , and blood volume and 
 velocity may be different from those of patients without anesthesia . the pharmacological effects of anesthetics itself can influence the 
 tic values . in a previous study , the 
 vasodilation effect of anesthetics altered local blood pressure and heart rate , and tp in 
 liver was about 11.6 sec faster in dogs without anesthesia than for those under anesthesia 
 . second , it was difficult to assess blood 
 perfusion accurately in some muscular areas containing fascia , ligaments and tendons , 
 because they show hyperintensity . although there were some limitations , ceus is expected to be an excellent modality for 
 evaluating changes of blood perfusion in skeletal muscle . this study provided quantitative 
 reference value of muscular perfusion including blood volume and velocity in normal dogs . a 
 0.5 ml aliquot contrast medium is suggested as a practical dose for 
 assessing the skeletal muscle . the ceus protocol for evaluating the blood perfusion of 
 skeletal muscles can be used as basic information for further studies evaluating the blood 
 perfusion of skeletal muscles in various pathologic states including bone fracture using 
 ceus .
this study evaluated perfusion of skeletal muscle using contrast enhanced ultrasonography 
 in humerus , radius , femur and tibia in normal dogs . 
 contrast enhanced ultrasonography for 
 each region was performed after injecting 0.5 ml and 1 ml of contrast medium ( sonovue ) in 
 every dog . 
 blood perfusion was assessed quantitatively by measuring the peak intensity , 
 time to the peak intensity and area under the curve from the time 
 intensity curve . 
 vascularization in skeletal muscle was qualitatively graded with a score of 03 according 
 to the number of vascular signals . a parabolic shape of time 
 intensity curve was observed 
 from muscles in normal dogs , and time to the peak intensity , the peak intensity and area 
 under the curve of each muscle were not significantly different according to the 
 appendicular regions examined and the dosage of contrast agent administered . 
 this study 
 reports that feasibility of contrast enhanced ultrasonography for assessment of the 
 muscular perfusion in canine appendicular regions .