Journal of Veterinary Cardiology (2007) 9, 15e24 www.elsevier.com/locate/jvc Contrast echocardiography in Boxer dogs with and without aortic stenosis Katja Höglund, DVM a,*, Claudio Bussadori, DVM, MD, Dipl ECVIM b, Oriol Domenech, DVM b, Jens Häggström, DVM, Dipl ECVIM c, Danitza Pradelli, DVM b, Clarence Kvart, DVM, Dipl ECVIM a a Department of Anatomy and Physiology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, PO Box 7011, 750 07 Uppsala, Sweden b Clinica Veterinaria Gran Sasso, Via Donatello 26, 201 31 Milano, Italy c Department of Clinical Sciences, Small Animals, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, PO Box 7037, 750 07 Uppsala, Sweden Received 29 June 2005; received in revised form 14 January 2006; accepted 18 February 2006 KEYWORDS Contrast echocardiography; Doppler; Phonocardiography; Aortic stenosis; Canine Abstract Objectives: The aim of this study was to investigate whether contrast echocardiography could enhance the subcostal Doppler signal for aortic flow measurements and achieve myocardial opacification, in Boxer dogs with and without AS. Background: In evaluating dogs for aortic stenosis (AS) subcostal Doppler echocardiography was used for measurement of the aortic flow velocity, a measurement that can sometimes be difficult to perform in Boxer dogs. Animals, materials, and methods: Cardiac auscultation, phonocardiographic and echocardiographic examinations, including a contrast study with Optison, were performed on 29 Boxer dogs selected based on previous examinations. Results: The initial subcostal Doppler signal was weak in 66% of the dogs and a marked improvement was seen in all dogs after contrast injection. The peak aortic flow velocity increased 5% from 2.58  1.42 m/s before contrast to 2.71  1.54 m/s after contrast (p ¼ 0.003). This corresponds to a 2.8 mmHg increase in the pressure gradient from 26.6 mmHg before to 29.4 mmHg after contrast. A dose of 0.05e0.1 mL of Optison administered intravenously resulted in approximately 4 min of Doppler signal enhancement. With the present technique contrast echocardiography did not achieve myocardial opacification. * Corresponding author. E-mail address: katja.hoglund@afys.slu.se (K. Höglund). 1760-2734/$ - see front matter ª 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jvc.2006.02.003 16 K. Höglund et al. Conclusions: Single use of the contrast agent Optison can be recommended for enhancement of the subcostal Doppler signal in dogs, in which plain Doppler signals are difficult to obtain. Albeit statistically significant, the mild increase in peak aortic flow velocity after contrast was not considered biologically or clinically significant. ª 2007 Elsevier B.V. All rights reserved. Introduction Aortic stenosis is a common disease in the Boxer breed.1e4 Physical examination of dogs affected by the disease reveals a systolic left basilar ejection murmur, which can be detected by cardiac auscultation and documented by phonocardiographic examination. The diagnosis is confirmed by echocardiographic examination. The subvalvular aortic ridge can usually be identified by an experienced echocardiographer and mildly thickened aortic valves can sometimes be visualized.5 Additionally, mild aortic hypoplasia associated with turbulent and elevated aortic blood flow velocities, appears to occur in the Boxer breed.6 Spectral Doppler echocardiography is used for measurement of the aortic flow velocity, which is often increased in dogs with AS.2,5e8 The subcostal transducer location is recommended for measurement of the aortic flow velocity.9 However, this measurement may be difficult to perform in some dogs due to the long distance from the transducer to the point of measurement. In Boxer dogs, this distance is around 20 cm. In addition, Boxer dogs have narrow, deep chests and sub-optimally positioned hearts, rendering the examination even more difficult. A panting dog may complicate the situation even further. Doppler tracings may thus become weak, and sometimes immeasurable, in these dogs. Recently, there has been a rapid development in the area of contrast echocardiography. Microbubble based sonographic contrast agents act by increasing ultrasound scattering and thereby enhancing ultrasound images and Doppler signals. The ideal ultrasound contrast agent is a non-toxic intravenous injectable, which is capable of passing through the pulmonary, cardiac and capillary vascular beds and is stable for recirculation.10 These criteria are met by Optison, a second generation contrast agent composed of octafluoropropane-filled human albumin microspheres.11 Optison has been proven to achieve myocardial opacification and is capable of enhancing Doppler ultrasound signals, in humans as well as in dogs.12e17 Its hemodynamic characteristics, efficiency and safety have been investigated in dogs,13e14,18 but, to our knowledge, there are no published studies on contrast Doppler investigations in dogs with aortic stenosis. Our hypothesis was that the use of a contrast agent would enhance the subcostal Doppler signal and achieve myocardial opacification, thereby facilitating the echocardiographic evaluation of dogs suspected of having AS. The aims of this study were therefore to investigate whether contrast echocardiography, using a single intravenous injection of Optison, could enhance the subcostal Doppler signal and achieve myocardial opacification in Boxer dogs with or without aortic stenosis, and if so, what would be the recommendable contrast dose and the duration of the effect. Animals, materials and methods General protocol Twenty-nine privately owned Boxer dogs, 13 females and 16 males, aged 3.0  2.3 years (range 0.7e10.5 years), were examined at Clinica Veterinaria Gran Sasso in Milano, Italy. The dogs were selected based on previous examinations at the clinic, ensuring a range of heart murmurs as well as echocardiographic findings. The examiners involved in the study were unaware of previous examination results. Upon arrival, each dog was weighed and the nutritional status was assessed. An identification protocol was filled out and written consent for the study was obtained from the dog owners. For characterization purposes, each dog underwent cardiac auscultation and a phonocardiographic examination. Thereafter, an intravenous catheter was placed in the cephalic vein and a complete echocardiographic examination, including aortic Doppler flow measurements before and after an intravenous contrast injection, was performed on each dog. To eliminate the risk of anaphylactic reactions, all owners were instructed to avoid future examinations with agents containing human albumin on their dogs. In order to monitor the health status of the dogs, a follow-up letter was sent to all dog owners 6 months after the contrast study. Contrast echocardiography in Boxer dogs Cardiac auscultation and phonocardiographic examination The cardiac auscultation and phonocardiographic examination both took place in a quiet examination room with the dog in a standing position. Initially, all dogs were auscultated by two examiners (KH, CK), using membrane stethoscopes.d The degree of heart murmur (scale 0e6) was decided by taking the average of the grades assigned by two separately blinded observers. If the average grade was not a whole number, the figure was rounded down to the closest whole number. Auscultation was followed by phonocardiographic examination. Phonocardiograms (PCG) were recorded using a piezo-electric phonocardiographic microphonee connected to a Siemens Mingograph Minor 3/Phonof amplifier as described by Häggström et al.19 The microphone was applied to the left side of the thorax over the aortic area and kept in this position during each study. All PCG recordings were later evaluated blindly by one examiner (KH), according to a protocol previously described.19,20 The dogs were divided into five groups (0e4) according to the severity of the phonocardiographic changes.21 Group 0 was defined as dogs with no murmur. Group 1 was defined as dogs with short, early systolic murmurs with a maximal duration of 50% of systole and with only high frequency components. Group 2 was defined as dogs with early to mid systolic murmurs with durations of 50e85% of systole and only high frequency components. Group 3 was defined as dogs with early to mid systolic murmurs with durations of 50e85% of systole and medium and high frequency components. Group 4 was defined as dogs with murmurs with durations of 85e100% of systole demonstrating high, medium and low frequency components. Contrast agent Optisong is a second-generation echocardiographic contrast agent. It consists of octafluoropropanecontaining microspheres of heat treated human albumin, suspended in a 1% human albumin solution. Each milliliter of Optison contains 5e8  108 d Rappaport-Sprague, Supraphone, Wilhelm Hasselmeyer Medical Development Invest. GmBH, Buchen, Germany. e Hellige GmbH, Heinrich-von-Stephanstrasse 4D, 7800 Freiburg, Germany. f Siemens Mingograph Minor 3/Phono, Siemens-Elema AB, Solna, Sweden. g Optison, Mallinckrodt Medical GmbH, Postfach 1462, 53761 Hennef, Germany. 17 microspheres with a mean diameter of 2.0e 4.5 mm, of which 93% are <10 mm in diameter. Before intravenous injection simple re-suspension of the microspheres, by gentle rotation of the vial, is required. Echocardiographic examination and contrast study The echocardiographic examinations took place in a quiet room and were performed by a veterinarian with extensive experience in canine echocardiography (CB). The echocardiographer did not auscultate the dogs and had no access to auscultatory or phonocardiographic results. An Esaote Biomedica Megas 449Xa Plus Multigraph ultrasound system,h with a 2.5e3.5 MHz phased array transducer, was used. For CW Doppler a 2.5e3.5 MHz annular array was used. Continuous ECG monitoring was used on all dogs and the examinations were recorded on super VHS videotapes. Gain and transmit power settings were optimized for individual dogs at the beginning of each study for 2D and Doppler echocardiography, and were not changed during the remainder of the protocol. A complete echocardiographic examination with standardized imaging planes22 was performed, with special attention to 2D changes indicative of aortic or pulmonic stenosis. Pulsed wave Doppler was used for measurement of pulmonic flow velocity. Aortic flow velocity was measured by continuous wave Doppler using the subcostal transducer location recommended by Lehmkuhl and Bonagura.9 Initially this measurement was performed without contrast, and then with the transducer kept in the same position, an intravenous injection of Optison was given. The contrast dose was flushed into the circulation with 5 mL of saline. Different doses of Optison were evaluated. Before our investigation a pilot study was performed wherein two dogs were injected with 0.5 mL of Optison each, i.e. the dose recommended by the manufacturer. In both dogs, massive saturation artifacts were observed. Based on this observation, we decided to investigate doses lower than 0.5 mL by using two dose groups. The first group consisted of 18 dogs, which each were given 0.2 mL IV, corresponding to 3.4e 5.5  106 octafluoropropane-containing microspheres of heat treated human albumin per kg bodyweight. The second group consisted of 11 dogs, which each were given 0.025e0.1 mL IV, h Esaote Biomedica Megas 449Xa Plus Multigraph ultrasound system, Esaote Biomedica, Via di Caciolle, Firenze, Italy. 18 corresponding to 4.4e28  105 octafluoropropanecontaining microspheres of heat treated human albumin per kilogram bodyweight. Measurements and evaluation of echocardiographic examination and contrast study M-mode values of the left ventricle were measured routinely. The size of the aorta and left atrium were measured on the 2D echocardiogram, according to Hansson et al.23 The aortic flow velocity before and after contrast, as well as the pulmonic flow velocity, were measured from three consecutive beats and the means were calculated. The subcostal Doppler signal was evaluated qualitatively before and after contrast. Before contrast the signal was judged as weak (faint Doppler tracing not seen in its entirety), intermediate (Doppler envelope identifiable, but suboptimal) or good (clear Doppler envelope). After contrast, a score of 0e3 was given each dog, where: 0, no signal enhancement; 1, slight signal enhancement, but spectral trace not seen in its entirety; 2, optimal signal enhancement with spectral trace demonstrated in its entirety; and 3, excessive signal enhancement with saturation artifacts present, mainly mirror artifacts across the x-axis (modified after Albrecht et al.24). The heart rate at the time of Doppler signal evaluation, before and after contrast, was calculated. The duration of contrast enhancement was measured during the investigation, in 17 of the dogs. In 14 of these dogs the measurements were later repeated from the tape recordings. During the second round of measurements the observer was blinded to the results from the first round. The 2D myocardial image quality was assessed by three independent observers, where one was non-blinded and two were blinded. The assessments were performed before and after contrast, using the following scores of image quality; poor, intermediate or good. K. Höglund et al. association between 2D visible AS and peak aortic flow velocity, before and after contrast, was evaluated using Wilcoxon signed-rank test. The difference in mean aortic velocity, as well as the difference in heart rate, before and after contrast, was evaluated using Wilcoxon signed-rank test. The difference in bodyweight between dose groups was evaluated using the ANOVA t-test. The association between auscultatory murmur grade and aortic flow velocity, in dogs with no visible AS on the 2D echocardiogram, before and after contrast, was evaluated using a multiple comparison t-test (TukeyeKramer), after equal variances between the groups was confirmed by the F-test. The difference in duration of contrast enhancement between two separate measurements, as well as the difference in 2D myocardial image quality before and after contrast, was evaluated using Wilcoxon signed-rank test. Inter-observer variation was investigated using weighted kappa-analysis. Results are reported as mean  SD. Level of significance was set at p < 0.05. Results Auscultation and phonocardiography Six dogs were found free of heart murmur on cardiac auscultation. The remaining 23 dogs all had varying degrees of systolic heart murmurs with their point of maximal intensity over the aortic area. Five dogs had a grade 1/6 murmur, 11 dogs grade 2/6, 3 dogs grade 3/6, 3 dogs grade 4/6 and 1 dog grade 6/6. On phonocardiographic examination 4 dogs were found free of heart murmur, while 3 dogs were assigned to PCG group 1, 15 dogs to group 2, 4 dogs to group 3 and 3 dogs to group 4. Diastolic murmurs were seen in 3 dogs on PCG. These dogs belonged to the systolic PCG groups 2, 3 and 4, respectively. Echocardiographic examination and contrast study Statistical analyses The results were evaluated using a computerized statistical program, JMP 4.0.2.i The differences in 2D diastolic echocardiographic parameters (IVSd, PWd, LVDd and LA/Ao), as well as in FS, between dogs with and without 2D visible AS were evaluated using the ANOVA t-test. The i JMP 4.0.2, SAS Institute Inc., Cary, NC, USA. The mean pulmonic flow velocity was 1.13  0.21 m/s and no morphological changes indicative of pulmonic stenosis were observed. Structural evidence of aortic stenosis (hereafter termed 2D visible AS) was identified in 7 (4 males and 3 females) of the 29 dogs (24%) on 2D echocardiogram. Of these 7 dogs, 5 had a subvalvular aortic ridge and 2 dogs had mildly thickened aortic valves. Echocardiographic 2D data were compared between dogs with 2D visible AS, and dogs without. The diastolic Contrast echocardiography in Boxer dogs 19 septal (IVSd) and posterior wall (PWd) thicknesses were both significantly increased in dogs with 2D visible AS. There was, however, no significant increase in diastolic left ventricular diameter (LVDd), fractional shortening (FS) or left atrial/aortic ratio (LA/Ao) in dogs with 2D visible stenosis, compared to dogs without (Table 1). In dogs with 2D visible AS the aortic flow velocity was 4.53  1.59 m/s (range 2.45e6.78 m/s) before contrast and 5.02  1.64 m/s (range 2.57e7.11 m/s) after contrast. In dogs with no 2D visible AS, the aortic flow velocity was 1.90  0.30 m/s (range 1.19e2.42 m/s) before contrast and 1.98  0.24 m/s (range 1.49e2.35 m/s) after contrast. The aortic flow velocity was significantly increased in dogs with 2D visible AS, both before and after contrast (p < 0.001), compared to dogs with no 2D visible AS. When dogs with 2D visible AS were excluded, there was no correlation between auscultatory murmur grade and aortic flow velocity before contrast. However, after contrast, the aortic flow velocity was significantly increased in dogs with a grade two murmur compared to dogs with no murmur (Fig. 1). The initial subcostal signal was judged as weak in 19 of the 29 dogs (66%), intermediate in 5 dogs and good in 5 dogs. In 2 of the 19 dogs (7%) with weak signals the tracings were immeasurable whereas the other 17 velocity spectra were measurable although potentially underestimated. Signal enhancement after contrast was seen in all dogs (Fig. 2). Hence, peak aortic flow velocity was measured in 27 dogs (93%) before contrast and in all dogs after contrast. The aortic flow velocity increased in 74% of the dogs after contrast. When comparing the peak aortic flow velocity in all dogs, there was a significant 5% increase from 2.58  1.42 m/s before contrast to 2.71  1.54 m/s after contrast (p ¼ 0.003). There Table 1 Diastolic echocardiographic parameters and fractional shortening in dogs with 2D visible aortic stenosis (n ¼ 7) and dogs without 2D visible aortic stenosis (n ¼ 22), including p-values Dogs with 2D visible AS IVSd (cm) 1.05  0.19 PWd (cm) 1.09  0.22 LVDd (cm) 4.48  0.61 FS (%) 32.14  7.99 LA/Ao 1.71  0.10 Dogs without 2D p-value visible AS 0.83  0.15 0.90  0.17 4.58  0.43 28.41  5.59 1.55  0.05 0.004 0.020 0.922 0.177 0.163 AS, aortic stenosis; IVSd, diastolic interventricular septum; PWd, diastolic posterior wall; LVDd, diastolic left ventricular diameter; FS, fractional shortening; LA/Ao, left atrial/aortic ratio. was no significant difference in aortic flow velocity before (p ¼ 0.59) or after contrast (p ¼ 0.53) between the high and low dose groups. There was no significant difference between mean heart rates before (86  20 beats/min) and after contrast (88  20 beats/min), (p ¼ 0.90), when looking at all dogs taken together. Similarly there was no difference in mean heart rate before (p ¼ 0.17) or after contrast (p ¼ 0.14) between the two dose groups. The mean bodyweight was 29.3 kg in the first group and 28.5 kg in the second group (p ¼ 0.66). In the dogs receiving 0.2 mL of Optison 56% were given score 2 (optimal signal enhancement) and 44% score 3 (excessive signal enhancement). In the second group of dogs, receiving 0.025e0.1 mL of Optison, 18% were given score 1 (slight signal enhancement), 64% were given score 2 and 18% score 3. Hence, the group of dogs with the highest percentage of Doppler examinations given score 2 (optimal signal enhancement), was the one given 0.025e0.1 mL of Optison. The mean duration of Doppler signal enhancement was 4.5 min (range 3e5.5 min) in the group of dogs given 0.2 mL of Optison and 4 min (range 2e4.5 min) in the group given 0.025e0.1 mL of Optison. The durations were measured twice, with a tendency towards longer duration during the first round of measurements, compared to the second round (p ¼ 0.09). The difference was, however, not significant. The 2D myocardial image quality, before contrast, was judged as good in 46%, intermediate in 53% and poor in 1% of the observations made by the three independent observers. The inter-observer agreement between the two blinded observers was good,25 with a weighted kappa-value of 0.67. When comparing the assessments made before and after contrast, there were no significant differences in 2D myocardial image quality when looking at all dogs together or between the two dose groups, identified by any of the three observers. No acute adverse side effects were seen during Optison application or within several hours thereafter. The questionnaire, which was sent to the dog owners 6 months after the study, was answered by all owners, except one. One dog had died due to gastric dilatation volvulus. The remaining dogs were all alive and none had shown signs of any disease obvious to the owners. Discussion Echocardiographic contrast agents have been proven to optimize both Doppler and grey-scale 20 K. Höglund et al. Figure 1 Aortic flow velocities (A) before and (B) after contrast, compared to auscultatory murmur grades, in dogs with no visible aortic stenosis on 2D echocardiogram. The mean aortic flow velocity for the six dogs without a cardiac murmur was 1.66  0.28 m/s before and 1.80  0.24 m/s, after contrast. Five dogs had a grade 1/6 murmur with mean aortic flow velocities of 2.01  0.29 m/s before, and 1.97  0.16 m/s, after contrast. In two of the dogs with grade 2/6 murmurs the aortic flow velocity was immeasurable before contrast. Hence, the number of dogs with grade 2/6 murmurs was 9 before and 11 after contrast. In these dogs, the mean aortic flow velocities before and after contrast were 2.01  0.24 and 2.08  0.23 m/s, respectively. There was no significant difference between murmur grades before contrast (A), whereas dogs with grade 2/6 murmurs had significantly higher aortic flow velocities, than dogs with no murmur, after contrast (B). images.26 The focus of the current study was on Doppler examination of aortic flow velocity. The measurements were made from the subcostal view, a position known to be difficult in some Boxer dogs. In two-thirds of the examined dogs Doppler signals before contrast were weak. This caused poor definition of the graph margins, which resulted in uncertainty of some of the measurements. In two of the dogs, the measurements were impossible to perform. In all of these dogs a marked enhancement of the Doppler signal was seen after contrast injection and the two immeasurable readings became measurable. However, considering the fast development within the area of diagnostic imaging, newer and future generation ultrasound equipment with improved Doppler capacity might result in lower percentage of weak signals. The dose group with the highest percentage of optimal Doppler signal enhancements was 0.025e0.1 mL. However, due to the difficulties in administering a dose as low as 0.025 mL, a more appropriate dose for clinical use, in adult Boxer dogs, is 0.05e0.1 mL of Optison. The duration of Doppler signal enhancement in the lower dose group (0.025e0.1 mL) was slightly shorter than in the higher dose group (0.2 mL), but with the lower dose measurements could be made instantly, making the examination quick and efficient, especially important in difficult, restless, patients. When Figure 2 Aortic flow measurement by subcostal continuous wave Doppler in a dog with a weak initial signal (A) and the same dog after injection of 0.05 mL of Optison (B). The peak aortic flow velocity is 4.5 m/s, as measured in image B. In this image aortic insufficiency can also be seen. The time lapse in each image is 2.5 s. Both images were obtained with a 2.5e3.5 MHz annular array transducer. Contrast echocardiography in Boxer dogs using the higher dose there was a high percentage of saturation artefacts, which meant having to wait for 1e1.5 min before measurements could be made, obviously inconvenient in the clinical situation. As expected, the peak aortic velocity was significantly increased in dogs with visible AS on 2D echocardiography before contrast, compared to dogs with no 2D visible AS. Similarly, the peak aortic velocity after contrast was significantly higher in dogs with 2D visible AS, compared to those without stenosis. Additionally, the peak aortic velocity was significantly increased after contrast, compared to before contrast, in all but seven dogs. Neither Nakatani et al.,27 nor von Bibra et al.28 found increased peak velocities across the aortic valve after contrast in humans with high quality baseline studies. However, several recent studies in human patients with aortic stenosis, have found higher peak aortic velocities measured by contrast Doppler, compared to conventional Doppler.29e31 By way of example, Smith et al.30 found a contrast-driven elevation of peak flow velocity of 8e12% and Almeida et al. found a 15% difference in peak flow velocity after contrast.29 In a study by Pugh et al.32 elevations in peak flow velocities were observed in the abdominal aortas of three healthy beagle dogs, after injection of the ultrasonographic contrast agent EchoGen. Strauss et al.33 studied flow velocities in the renal and iliac arteries of dogs and found an increase in the recorded maximum Doppler flow velocities of 25e30% after injection of the contrast agent BY963. They attributed this to the amplification of all components of the blood flow profile following echo contrast application, and specifically emphasized the importance of enhancing the relatively small number of erythrocytes flowing at the highest velocity in the arteries, which are likely to disappear in noise if they are not enhanced by a contrast agent. A similar situation is likely to occur with the use of Optison as a contrast enhancer. We did not find a significant change in heart rate, in our study. Similarly, Mobarek et al.12 found no significant changes in heart rate, mean arterial blood pressure or left atrial pressure when studying Optison in dogs. Accordingly, the increase in peak aortic velocity should not be caused by a hypothetical positive inotropic or chronotropic effect induced by the contrast agent, or by stress. However, direct physiological stimuli by the contrast material, causing higher pressure gradients by an increase in myocardial force of contraction and/or decrease in after-load, can not be entirely ruled out. If they occur simultaneously, they may result in increased 21 forward flow in terms of volume, without necessarily affecting blood pressure or heart rate. Since our hemodynamic monitoring can not rule out this possibility, it should be seen as a potential study limitation. Albeit statistically significant, the increase in peak aortic flow velocity of 5% in our study is not considered biologically significant. When translating the mean aortic flow velocities before and after contrast into pressure gradients (26.6 and 29.4 mmHg, respectively, i.e. an increase by 2.8 mmHg), it becomes apparent that this mild increase is clinically insignificant for an individual dog. The upper limit of normal aortic flow velocity is controversial. Even though most cardiologists agree that velocities greater than 2 m/s are supportive of the diagnosis, elevated aortic flow velocity alone should not be used for diagnosis of AS.5 For optimal diagnostic results, the aortic flow measurement should be combined with other echocardiographic findings and clinical data, such as murmur characteristics from cardiac auscultation and phonocardiographic examination, structural evidence of the stenotic lesion on 2D echocardiogram and identification of turbulence in the left ventricular outflow tract by PW or CFM Doppler.2,5e6 Stress has been proven to increase the aortic flow velocity in dogs with AS.34 Thus the degree of stress during the echocardiographic Doppler examination should be assessed and incorporated into the diagnostic evaluation of the individual dog. Additionally, one should bear in mind that aortic peak flow velocities may be slightly, but significantly, higher when using Optison as a Doppler signal enhancer. Interestingly, when excluding dogs with visible AS on 2D echocardiogram, the aortic flow velocity after contrast was significantly higher in dogs with grade two murmurs on cardiac auscultation, compared to dogs with no murmur. This difference was not seen before contrast. Several of the grade two dogs had aortic flow velocities greater than 2 m/s. Hence, there seems to be a difference in aortic flow velocity even in dogs with no visible aortic stenosis on the 2D echocardiogram, a difference that might be explained by the possible existence of aortic hypoplasia in the Boxer breed6 with a different hemodynamic pattern. Similar findings were made in a retrospective study of 201 Boxer dogs without overt structural evidence of AS on 2D echocardiography and murmur grades from 0e3.35 Dogs with murmurs had significantly higher aortic flow velocities than dogs without murmurs. This was shown with conventional Doppler, whereas in our study significant differences were shown only after contrast injection. However, since two 22 of the dogs in our material (both of which had grade 2/6 cardiac murmurs) gave initial subcostal tracings which were immeasurable, the comparison between murmur groups was performed with a lower number of dogs before contrast. With a higher number of dogs, it is plausible that a significant difference between dogs with no murmur and dogs with grade 2/6 murmurs would have also been shown before contrast. The disparity between the two studies might also be explained by the different murmur grades included, i.e. murmur grades 0e2 in our study, compared to grades 0e3 in the retrospective study. However, the importance of the elevated aortic flow velocity in dogs with low grade murmurs but no 2D echocardiographic structural left ventricular outflow tract obstruction and their possible connection to AS remains unclear. Further studies are needed to bring more clarity to this question. An important indication for contrast echocardiography in humans with suspected coronary artery disease,11 and a possible indication for dogs with aortic stenosis, is the assessment of myocardial perfusion. The anatomy as well as the position of the canine heart differs from the human heart. Humans have flat chests, whereas dogs, especially Boxer dogs, have narrow, deep chests. Furthermore, obesity is a well-known factor lowering the quality of echocardiographic images in humans, whereas this problem is less common when examining dogs. These factors contribute to higher quality 2D base-line echocardiographic images in most dogs, compared to human cardiac patients. This might be one reason why we did not find an improvement in 2D myocardial image quality post contrast injection, and hence did not achieve myocardial opacification, as opposed to previous results.12e14 Another possible reason for this lack of improvement is the study design, which focused on the Doppler examination. During contrast injection and the first minutes thereafter Doppler recordings of the aortic flow were made. Thereafter the myocardium was studied. Since contrast duration of Optison is 2e5 min,15 which is in accordance with our findings, most of the contrast would be gone when myocardial studies were performed. Additionally, our contrast doses were optimized for Doppler investigation and are lower than optimal doses previously described for myocardial opacification in dogs (varying between 0.1e0.7 mL).12e14 Hence, it is possible that the dose needed for myocardial opacification is higher than the optimal dose for Doppler assessment of large arteries, which would further explain our lack of improvement in 2D myocardial image quality. K. Höglund et al. In dogs affected by AS, the increased resistance to left ventricular systolic outflow increases the wall stress, which stimulates an increase in left ventricular muscle mass.5 In the present study, concentric left ventricular hypertrophy with increased diastolic septal and posterior wall thicknesses were demonstrated in dogs with 2D visible AS, while the left ventricular fractional shortening was normal, a common finding in dogs affected by AS.5 Four of the dogs in our study can be considered severely affected by aortic stenosis, with peak aortic flow velocities exceeding 4.5 m/s, corresponding to peak pressure gradients above 80 mmHg. In these dogs, the myocardial viability can be highly compromised by the synergistic role of increased myocardial oxygen consumption and the decreased coronary perfusion pressure. As discussed above, none of these dogs or any of the other dogs, however, demonstrated increased myocardial echo-densities following contrast injection. This precludes our ability to conclude that myocardial perfusion is changed either by disease or by contrast injection in our study population. In addition, the decreased coronary perfusion pressure is mainly due to decreased diastolic pressure at the coronary artery ostia, because they are located distal (downstream) to the stenotic lesion-level. Hypertrophy (if severe enough) may contribute further to the compromise in myocardial viability, if it overwhelms the ability of coronary capillary endothelial proliferation and collateralization, thereby challenging viability by increasing the oxygen delivery distance (ODD) required to supply hypertrophied myocardial territory between coronary capillaries. This would further explain our lack of demonstrated myocardial opacification following contrast injection in the dogs that had aortic stenosis. Since Optison contains human serum albumin there is a possibility of adverse anaphylactic reactions due to antibody formation, if it is used repeatedly in dogs. At the time of our study there were no reported cases of anaphylactic reactions in dogs. Since then, however, Yamaya et al.18 have reported that two dogs developed severe systemic hypotension following Optison injections. The hypotension was reproduced with 1 mL of albumin, excluding other components of Optison. The doses given in that study (1, 3 and 5 mL of Optison sequentially) were much higher than our maximum dose of 0.2 mL. Additionally, Meza et al.13 gave 222 injections of Optison to 7 dogs, with a maximal cumulative dose of 37 mL, with no adverse side effects. The risk of adverse anaphylactic reactions could, however, not be excluded. In our consent form the dog owners were instructed to avoid Contrast echocardiography in Boxer dogs similar contrast examinations in their dog in order to avoid any risk of anaphylactic reactions. No acute adverse side effects were seen during Optison application or within several hours thereafter. Likewise, no unexpected long-term clinical side effects were detected by the questionnaire answered by the dog owners 6 months after participation in our study. However, future use of contrast echocardiography in dogs should ideally be performed with contrast media not containing human serum albumin. Recommendations  Contrast echocardiography can be used as an enhancer of the subcostal Doppler signals in Boxer dogs, in which plain Doppler signals are difficult to obtain.  The recommended dose of Optison for clinical use in adult Boxer dogs is 0.05e0.1 mL intravenously, allowing contrast duration of up to 4 min.  Myocardial opacification was not achieved following contrast injection. This indication can therefore not be recommended with the present technique. Acknowledgments Funding for this study was provided by Agria Insurance Company, Stockholm, Sweden. Preliminary data was presented at the 13th ECVIM-CA Congress, Uppsala, Sweden, September 4e6, 2003. References 1. O’Grady MR, Holmberg DL, Miller CW, et al. Canine congenital aortic stenosis: a review of the literature and commentary. Can Vet J 1989;30:811e5. 2. Bussadori C, Quintavalla C, Capelli A. Prevalence of congenital heart disease in boxers in Italy. J Vet Cardiol 2001;3:7e11. 3. Tidholm A. Retrospective study of congenital heart defects in 151 dogs. J Small Anim Pract 1997;38:94e8. 4. Fuentes VL. Aortic stenosis in boxers. In: Raw TPME, editor. The veterinary annual. 33rd ed. London: Blackwell Scientific; 1993. p. 220e9. 5. Kienle RD. Aortic stenosis. In: Kittleson MD, Kienle RD, editors. Small animal cardiovascular medicine. St. Louis, MO: Mosby; 1998. p. 260e72. 6. Bussadori C, Amberger C, Le Bobinnec G, et al. Guidelines for the echocardiographic studies of suspected subaortic and pulmonic stenosis. J Vet Cardiol 2000;2:17e24. 7. Lehmkuhl LB, Bonagura JD. CVT update: canine subvalvular aortic stenosis. In: Bonagura JD, editor. Kirk’s current veterinary therapy XII. London. WB Saunders; 1995. p. 822e7. 8. Sisson DD, Thomas WP, Bonagura JD. Congenital heart disease. In: Ettinger SJ, Feldman EC, editors. Textbook of veterinary internal medicine: diseases of the dog and cat. Philadelphia, PA: WB Saunders; 2000. p. 737e87. 23 9. Lehmkuhl LB, Bonagura JD. Comparison of transducer placement sites for Doppler echocardiography in dogs with subaortic stenosis. Am J Vet Res 1994;55:192e8. 10. Goldberg BB, Liu JB, Forsberg F. Ultrasound contrast agents: a review. Ultrasound Med Biol 1994;20:319e33. 11. Clark LN, Dittrich HC. Cardiac imaging using Optison. Am J Cardiol 2000;86:14Ge8G. 12. Mobarek S, Kates M, Meza M, et al. Identification of perfusion abnormalities using FSO69, a novel contrast agent, in conscious dogs. Echocardiography 1997;14:337e44. 13. Meza M, Greener Y, Hunt R, et al. Myocardial contrast echocardiography: reliable, safe, and efficacious myocardial perfusion assessment after intravenous injections of a new echocardiographic contrast agent. Am Heart J 1996;132:871e81. 14. Skyba DM, Camarano G, Goodman NC, et al. Hemodynamic characteristics, myocardial kinetics and microvascular rheology of FS-069, a second-generation echocardiographic contrast agent capable of producing myocardial opacification from a venous injection. J Am Coll Cardiol 1996;28: 1292e300. 15. Cohen JL, Cheirif J, Segar DS, et al. Improved left ventricular endocardial border delineation and opacification with OPTISON (FS069), a new echocardiographic contrast agent. Results of a phase III Multicenter Trial. J Am Coll Cardiol 1998;32:746e52. 16. Schaffler GJ, Kugler CH, Schreyer G, et al. Quantitative and qualitative analysis of in vivo Doppler signal enhancement of FS-069. Invest Radiol 2002;37:1e6. 17. Brown JM, Quedens-Case C, Alderman JL, et al. Contrast enhanced sonography of visceral perfusion defects in dogs. J Ultrasound Med 1997;16:493e9. 18. Yamaya Y, Niizeki K, Kim J, et al. Effects of Optison on pulmonary gas exchange and hemodynamics. Ultrasound Med Biol 2002;28:1005e13. 19. Häggström J, Kvart C, Hansson K. Heart sounds and murmurs: changes related to severity of chronic valvular disease in the Cavalier King Charles spaniel. J Vet Intern Med 1995;9:75e85. 20. Heiene R, Kvart C, Indrebo A, et al. Prevalence of murmurs consistent with aortic stenosis among boxer dogs in Norway and Sweden. Vet Rec 2000;147:152e6. 21. Kvart C, French AT, Fuentes VL, et al. Analysis of murmur intensity, duration and frequency components in dogs with aortic stenosis. J Small Anim Pract 1998;39:318e24. 22. Thomas WP, Gaber CE, Jacobs GJ, et al. Recommendations for standards in transthoracic two-dimensional echocardiography in the dog and cat. Echocardiography Committee of the Specialty of Cardiology, American College of Veterinary Internal Medicine. J Vet Intern Med 1993;7:247e52. 23. Hansson K, Häggström J, Kvart C, et al. Left atrial to aortic root indices using two-dimensional and M-mode echocardiography in cavalier King Charles spaniels with and without left atrial enlargement. Vet Radiol Ultrasound 2002;43: 568e75. 24. Albrecht T, Urbank A, Mahler M, et al. Prolongation and optimization of Doppler enhancement with a microbubble US contrast agent by using continuous infusion: preliminary experience. Radiology 1998;207:339e47. 25. Altman DG. Inter-rater agreement. Practical statistics for medical research. London: Chapman and Hall; 1991. p. 403e9. 26. Strauss AL, Beller K-D. Persistent opacification of the left ventricle and myocardium with a new echo contrast agent. Ultrasound Med Biol 1999;25:763e9. 27. Nakatani S, Imanishi T, Terasawa A, et al. Clinical application of transpulmonary contrast-enhanced Doppler technique in 24 the assessment of severity of aortic stenosis. J Am Coll Cardiol 1992;20:973e8. 28. Von Bibra H, Sutherland G, Becher H, et al. Clinical evaluation of left heart Doppler contrast enhancement by a saccharide-based transpulmonary contrast agent. The Levovist Cardiac Working Group. J Am Coll Cardiol 1995; 25:500e8. 29. Almeida AG, Sargento L, Gabriel HM, et al. Evaluation of aortic stenosis severity: Role of contrast echocardiography in comparison with conventional echocardiography and cardiac catheterization. Rev Port Cardiol 2002;21: 555e72. 30. Smith LA, Cowell SJ, White AC, et al. Contrast agent increases Doppler velocities and improves reproducibility of aortic valve area measurements in patients with aortic stenosis. J Am Soc Echocardiogr 2004;17:247e52. K. Höglund et al. 31. Lipiec P, Plewka M, Krzeminska-Pakula M, et al. Usefulness of contrast enhancement for the echocardiographic quantitative assessment of aortic stenosis. Acta Cardiol 2004;59: 211e2. 32. Pugh CR, Arger PH, Sehgal CM. Power, spectral, and color flow Doppler enhancement by a new ultrasonographic contrast agent. J Ultrasound Med 1996;15:843e52. 33. Strauss AL, Beller K-D. Arterial parameters under echo contrast enhancement. Eur J Ultrasound 1997;5:31e8. 34. Höglund K, French A, Dukes-McEwan J, et al. Low intensity heart murmurs in boxer dogs: inter-observer variation and effects of stress testing. J Small Anim Pract 2004;45:178e85. 35. Koplitz SL, Meurs KM, Spier AW, et al. Aortic ejection velocity in healthy Boxers with soft cardiac murmurs and Boxers without cardiac murmurs: 201 cases (1997e2001). J Am Vet Med Assoc 2003;222:770e4.