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Quantitative Differences Between the First and Second Injection of Contrast Agent in Contrast-Enhanced Ultrasonography of Feline Kidneys and Spleen
Ultrasound in Med. & Biol., Vol. -, No. -, pp. 1–5, 2016 Copyright Ó 2016 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter
http://dx.doi.org/10.1016/j.ultrasmedbio.2016.09.013
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Original Contribution QUANTITATIVE DIFFERENCES BETWEEN THE FIRST AND SECOND INJECTION OF CONTRAST AGENT IN CONTRAST-ENHANCED ULTRASONOGRAPHY OF FELINE KIDNEYS AND SPLEEN EMMELIE STOCK,* KATRIEN VANDERPERREN,* HENDRIK HAERS,* LUC DUCHATEAU,y MYRIAM HESTA,z and JIMMY H. SAUNDERS* * Department of Veterinary Medical Imaging and Small Animal Orthopedics, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium; y Department of Comparative Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium; and z Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium (Received 31 May 2016; revised 8 September 2016; in final form 9 September 2016)
Abstract—Contrast-enhanced ultrasound is a valuable and safe technique for the evaluation of organ perfusion. Repeated injections of ultrasound contrast agent are often administered during the same imaging session. However, it remains unclear if quantitative differences are present between the consecutive microbubble injections. Therefore, the first and second injection of contrast agent for the left renal cortex, renal medulla and the splenic parenchyma in healthy cats were compared. A lower peak intensity and area under the curve were observed for the first injection of contrast agent in the feline kidney, both for the renal cortex and medulla, and spleen. Moreover, for the renal cortex, the time-intensity curve was steeper after the second injection. Findings from the present study demonstrate that a second injection of contrast agent provides stronger enhancement. The exact mechanism behind our findings remains unclear; however, saturation of the lung macrophages is believed to play an important role. (E-mail: [email protected]) Ó 2016 World Federation for Ultrasound in Medicine & Biology. Key Words: Contrast-enhanced ultrasound, Microbubbles, Multiple injections, Kidney, Spleen, Cat.
diffuse renal disorders, both kidneys must be imaged. Additionally, patient motion during the study may induce the need for multiple injections of contrast agent. Some authors already mentioned that tissue enhancement after the first injection of microbubbles is subjectively less bright in comparison with subsequent contrast agent injections (Haers et al. 2013; Pey et al. 2011; Salwei et al. 2005). A study on contrast-enhanced ultrasound of liver and aorta in human patients confirmed this finding quantitatively (Skrok, 2007). In contrast, no significant differences were found between the first and second injection of contrast agent in mice liver and tumor tissue. However, a tendency toward a higher peak intensity (PI) after consecutive microbubble injections was noted (Rix et al. 2014). Thus far, it remains unclear if there are significant differences between the first and second injection of contrast agent. Nevertheless, it is of major importance to know if differences are present between both injections, as this could influence study results. Therefore, the purpose of this study was to compare several perfusion parameters between the first and second injection in the left kidney and spleen in healthy cats. The
INTRODUCTION Contrast-enhanced ultrasonography allows real-time visualization of macro- and microvascularization. Creating time-intensity curves and calculating several perfusion parameters allows quantification of tissue perfusion. The technique is extremely safe and can be performed in patients with renal impairment (Piscaglia et al. 2006; Seiler et al. 2013). Contrast-enhanced ultrasonography shows great potential for evaluation and diagnosis of various renal disorders in human and veterinary medicine (Granata et al. 2015; Haers et al. 2013). Repeated injections of contrast agent are most often required in contrast-enhanced ultrasound studies for different reasons. The first injection is sometimes used to optimize image settings. Multiple lesions may also be present, necessitating several injections of contrast agent to allow study of all lesions. In the assessment of Address correspondence to: Emmelie Stock, Department of Veterinary Medical Imaging and Small Animal Orthopedics, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium. E-mail: [email protected] 1
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hypothesis is that the second injection of contrast agent will result in an increased enhancement of both the left kidney and the spleen; this will mainly be reflected by an increased PI and area under the curve (AUC). MATERIALS AND METHODS The experiments where approved by the Local Ethical Committee of Ghent University (EC2014/38). Seven healthy, purpose-bred laboratory cats with a mean age of 4.7 y and mean body weight of 3.1 kg were used. The cats were judged healthy based on physical examination, hematology, routine biochemistry, urinalysis and abdominal ultrasound. A 22-G catheter was placed in the cephalic vein. All cats were anesthetized with propofol (Propovet, Abbott Labs, Berkshire, UK), 6 mg/kg intravenous, maintained with additional boluses of 1 mg/kg on effect. A commercial contrast agent (Sonovue, Bracco, Milan, Italy) was administered at 0.15 mL per bolus, immediately followed by a 1-mL saline bolus. A three-way stopcock was used to avoid delay between the contrast agent and the saline flush. The same person performed the injections in a standardized manner. The cats received two consecutive injections, with approximately 10 min between the two injections. Between injections, the microbubbles were destroyed by setting the acoustic power at the highest value and scanning the caudal abdominal aorta, liver and spleen for approximately 2 min. A linear array transducer of 5–7.5 MHz on a dedicated ultrasound machine (MyLab 30CV, Esaote, Genoa, Italy), equipped with CnTI-contrast tuned imaging technology, was used. Machine settings were kept constant between the injections. A mechanical index of 0.09 was applied. The gain setting was adapted to a value just suppressing signals before arrival of contrast agent (55%– 58%). An image clip of 120 s at a frame rate 7.5 Hz was recorded for every injection. Imaging was started simultaneously with the contrast agent injection. The kidney was consistently imaged in a longitudinal plane. The spleen was imaged simultaneously with the left kidney. Quantitative analysis was performed using an offline image analysis program (Image J, US National Institutes of Health, Bethesda, MD, USA). The data were not linearized before quantification. Mean pixel intensity was measured every 1 s from 0 s to 70 s, every 2 s for 70 s to 90 s and every 4 s for 90 s to 120 s. A circular region of interest was drawn in the renal cortex and medulla at approximately the same location (ventral aspect of the renal cortex, central aspect of the renal medulla). The size of the region of interest was kept constant (1.2-cm diameter). A region of interest containing the complete visualized part of the spleen was drawn for the splenic pa-
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renchyma. Care was taken to avoid large vessels. The mean pixel intensity values were used to create timeintensity curves. The curves were analyzed for blood flow parameters representing blood volume (baseline intensity [BI], PI, AUC) and blood velocity (arrival time [AT], time-to-peak [TTP], wash-in/wash-out [Win/ Wout]). Wash-out was not calculated for the spleen, as only the first part of the wash-out occurred within the time the clip was captured (120 s). BI was defined as the intensity measured the first 3 s after contrast agent injection. AT was set at the point where the curve reached a value at least 5 mean pixel intensity values higher than BI. Wash-in was calculated from data points between 10% higher than BI and 90% of PI; wash-out from points between 90% of PI and T 5 120 s (end of the study). PI and AUC were corrected for the BI. Statistical test selection and analysis was performed by one of the authors (L.D.). The statistical analysis was based on a mixed model with the cat as the random effect and sequence (first or second injection) as the fixed effect, using F-tests at the 5% significance level (SAS Version 9.4, SAS Institute Inc, Cary, NC, USA). RESULTS AUC was significantly higher for the second injection compared to the first injection for both the renal cortex (p 5 0.01) and medulla (p 5 0.04). The PI was also higher for the second injection; this was only significant for the renal cortex (p 5 0.009) while a trend was present for the renal medulla (p 5 0.055). Additionally, for the renal cortex, the Win (p 5 0.01) and Wout (p 5 0.006) were less steep for the first injection compared to the second. No significant differences were present between the first and second injection for AT (cortex: p 5 0.4; medulla: p 5 0.3) and TTP (cortex: p 5 0.3; medulla: p 5 0.08). Similarly, the PI (p 5 0.007) and AUC (p 5 0.008) in the splenic parenchyma were significantly higher for the second injection compared to the first. The BI was consistently comparable for the first and second injection. Mean 6 standard error values of the renal blood flow parameters for the first and second injections are summarized in Table 1. Time-intensity curves comparing the first and second injection are represented by Figure 1. A contrast-enhanced ultrasound image of the left kidney and spleen of a cat is shown in Figure 2. DISCUSSION In the present study, significant differences were observed between the first and second injections of ultrasound contrast agent. The most reproducible differences between the two injections were a higher PI and AUC for the second injection, seen in the renal cortex, renal
Differences between the first and second injection of ultrasound contrast agent d E. STOCK et al.
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Table 1. Means and standard errors of perfusion parameters calculated from the renal cortex, renal medulla and splenic parenchyma in healthy cats, for the first and second injection of contrast agent Renal cortex Perfusion parameter BI PI AT TTP Win Wout AUC
Renal medulla
Spleen
First injection
Second injection
First injection
Second injection
First injection
Second injection
2.50 6 0.35 34.95 6 4.58* 7.51 6 0.61 41.75 6 2.36 1.17 6 0.19* 20.30 6 0,04* 2340.50 6 492.91*
2.96 6 0.35 51.13 6 4.58* 6.86 6 0.61 39.61 6 2.36 1.90 6 0.19* 20.43 6 0.04* 3769.82 6 492.91*
1.72 6 0.14 26.83 6 3.97 20.59 6 1.47 43.61 6 2.76 1.58 6 0.32 20.28 6 0.04 1077.03 6 322.53*
1.74 6 0.14 39.91 6 3.97 18.29 6 1.47 35.90 6 2.76 2.37 6 0.32 20.33 6 0.04 1983.53 6 322.53*
2.70 6 0.39 46.06 6 4.70* 7.22 6 0.55 61.03 6 1.29 1.74 6 0.45 3770.93 6 477.01*
3.13 6 0.39 59.60 6 4.70* 7.38 6 0.55 62.91 6 1.19 1.85 6 0.45 5235.02 6 477.01*
AT 5 arrival time; AUC 5 area under the curve; BI 5 baseline intensity; PI 5 peak intensity; TTP 5 time-to-peak; Win 5 wash-in; Wout 5 wash-out. * Value represents a significant (p , 0.05) effect.
medulla and splenic parenchyma. For the renal cortex, the wash-in and wash-out were also steeper after the second injection. In agreement with our results, a tendency toward a higher PI after consecutive microbubble injections was also noted in a recent study in mice tumors (Rix et al. 2014). However, in the study of Rix et al. (2014), the opposite effect, that is, a decreasing PI after consecutive injections, was observed in the liver. This might be due to short-term accumulation in liver Kupffer cells. In contrast, in a human study, an increased PI and AUC were noted in the liver after the second injection of microbubbles (Skrok, 2007).
Fig. 1. Mean time-intensity curves calculated from the renal cortex, renal medulla and spleen demonstrating a lower peak intensity and smaller area under the curve for the first injection of microbubbles. The time (in seconds) is displayed on the horizontal axis and the intensity (in arbitrary units [a.u.]) on the vertical axis.
There were no significant differences in BI between the first and second injection, indicating that no residual microbubbles were remaining in tissue of interest before the second injection. Between injections, the remaining microbubbles were destroyed by setting the acoustic power at the highest value and scanning the caudal abdominal aorta, liver and spleen for approximately 2 min. We avoided applying ultrasound waves of relatively high acoustic power on the kidney, as this could potentially cause capillary rupture (Miller et al. 2010; Williams et al. 2007). Rix et al. (2014) observed an increased heart rate after the first injection of microbubbles, which coincides with a faster circulation. This explains the faster washin. Moreover, an elevated heart rate and decreased TTP might result in a concentrated microbubble bolus, resulting in a higher PI. In humans, it was found that injections of relatively low volumes of phospholipid solutions might result in complement activation-related pseudo-allergy, causing a transient increase in heart rate and pulse pressure (Szebeni, 2011). The heart rate was not measured in our study. In another study by our research group, we did not observe a change in heart rate after consecutive contrast agent injections in healthy cats (unpublished results). Moreover, no effects on AT and TTP are present in the present study. It should be noted that the volume of the saline itself might cause an accelerated circulation in mice (Rix et al. 2014). In mice, short-term accumulation of microbubbles in the liver sinusoids and spleen might also explain the higher PI in tumors (Rix et al. 2014). It is unlikely that hepatic and splenic accumulation of contrast agent would explain the higher PI in the present study, as the majority of the microbubbles in the liver and spleen were destroyed by scanning these organs using high acoustic power. Moreover, it can be assumed that the low volume of contrast agent does not result in liver saturation in cats. In humans, injection of 1.2–1.4 mL microbubble solution does not result in liver saturation (Weskott, 2008). Another explanation for the stronger enhancement after the second injection might be saturation of the
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Fig. 2. Contrast-enhanced ultrasound images of the spleen and left kidney of a cat, illustrating a lower enhancement of after the first injection of contrast agent (left) compared to the second injection (right). Both images are obtained 30 s after contrast injection.
lung macrophages by the microbubbles (Skrok, 2007). This would allow more microbubbles to pass the lung and finally reach the systemic circulation. In pigs, administration of microbubbles resulted in an acute, transient and dose-dependent right ventricular dilation, increased pulmonary pressure and increased fractional shortening (Bramos et al. 2008). This could be caused by transient hypoxic pulmonary vasoconstriction. Pigs are known to develop severe pulmonary hypertension, which is likely to be caused by activation of pulmonary macrophages. Also, in dogs, increased pulmonary vascular resistance and pulmonary artery pressure was seen after administration of high doses of Levovist (Schwarz et al. 1996). No significant hemodynamic changes were observed at clinical, weight-adjusted doses in these dogs. The exact etiology of the effects on the pulmonary vascularization in these dogs remains unclear; transient pulmonary vasoconstriction might be a possible explanation (Schwarz et al. 1996). The last explanation that may be raised is a local effect caused by a vascular mediator. An interaction could be present between the contrast agent and the endothelium; moreover, the influence of the ultrasound waves on microbubbles could reinforce this effect. The combination of both could cause local activation of vasodilative substances. Wellperfused organs such as the kidneys and neoplastic tissues might therefore be more sensitive to this effect. Several factors may influence the presence and extent of differences between the first and second injection of contrast agent. First, the type of contrast agent may be an important factor. It is known that great differences are present in the extent and duration of phagocytosis by the reticuloendothelial system depending on the type of contrast agent (Tang et al. 2011). Generally, SonoVue shows a low uptake by Kupffer cells in contrast to other types of microbubbles, as do Sonazoid, Optison and Levovist (Yanagisawa et al. 2007). Moreover, species
may play an import role. As previously mentioned, pigs are prone to develop extensive pulmonary hypertension after activation of lung macrophages. To our knowledge, no information is available on this phenomenon in cats. Finally, the organ of interest may influence the results, as suggested in the murine study of Rix et al. (2014), which showed contrasting results in tumor tissue compared to the liver. In the latter study, peak enhancement in tumor tissue tended to increase after repeated microbubble injections, whereas the peak enhancement in the liver decreased steadily. Our study has some limitations. Cardiovascular parameters were not measured regularly throughout the study. However, data of other studies performed by our research group in feline patients revealed no significant changes in heart rate, blood pressure or respiratory rate after the first injection of contrast agent. The assessment of heart rate, blood pressure and even more interestingly, pulmonary artery pressure and the pressures in the different cardiac chambers, would deliver important information to distinguish between the possible causes for the low enhancement after the first injection. Body temperature was also not measured. It is known that hypothermia alters the blood flow and ultimately the distribution of contrast agent (Hyvelin et al. 2013). However, it is very unlikely that the cats would develop hypothermia, since the study duration was very short (15–20 min) and they were only superficially anesthetized. Moreover, only the first and second injections of contrast agent were studied. It would be interesting to have information about the subsequent injections of contrast agent. It remains unknown if differences are present between the second injection and following injections of contrast agent. Finally, we do not know how long this effect lasts. In other words, it is unclear if the same findings would be present if a second imaging session were performed in the same animal several hours later.
Differences between the first and second injection of ultrasound contrast agent d E. STOCK et al.
CONCLUSIONS We demonstrated clear differences are present between the first and second injection of contrast agent of the left kidney and spleen in cats. Less bright enhancement is achieved by the first injection. Unfortunately, we were not able to distinguish between the possible explanations for these findings. Activation/saturation of the lung macrophages is the most plausible explanation. Further research is necessary to clarify the mechanism of our findings and investigate if similar phenomena are present in other species and different organs. Our findings have important implications: the first injection should not be compared quantitatively to the second injection; additionally, use of a second injection is recommended for further analysis as it provides better enhancement. Acknowledgments—This research was supported by the Special Research Fund of Ghent University, Belgium (BOF grant 2015003301).
REFERENCES Bramos D, Tsirikos N, Kottis G, Pamboucas C, Kostopoulou V, Trika C, Ikonomidis I, Toumanidis S. The acute effect of an echo-contrast agent on right ventricular dimensions and contractility in pigs. J Cardiovasc Pharm 2008;51:86–91. Granata A, Zanoli L, Insalaco M, Valentino M, Pavlica P, Di Nicolo PP, Scuderi M, Fiorini F, Fatuzzo P, Bertolotto M. Contrast-enhanced ultrasound (CEUS) in nephrology: Has the time come for its widespread use? Clin Exp Nephrol 2015;19:606–615. Haers H, Daminet S, Smets PMY, Duchateau L, Aresu L, Saunders JH. Use of quantitative contrast-enhanced ultrasonography to detect diffuse renal changes in beagles with iatrogenic hypercortisolism. Am J Vet Res 2013;74:70–77. Hyvelin JM, Tardy I, Arbogast C, Costa M, Emmel P, Helbert A, Theraulaz M, Nunn AD, Tranquart F. Use of ultrasound contrast agent microbubbles in preclinical research recommendations for small animal imaging. Invest Radiol 2013;48:570–583.
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Miller DL, Dou CY, Wiggins RC. Contrast-enhanced diagnostic ultrasound causes renal tissue damage in a porcine model. J Ultras Med 2010;29:1391–1401. Pey P, Vignoli M, Haers H, Duchateau L, Rossi F, Saunders JH. Contrast-enhanced ultrasonography of the normal canine adrenal gland. Vet Radiol Ultrasound 2011;52:560–567. Piscaglia FB, Bolondi L, Italian Society for Ultrasound in Medicine and Biology (SIUMB) Study Group onUltrasound Contrast Agents. The safety of SonoVue in abdominal applications: Retrospective analysis of 23188 investigations. Ultrasound Med Biol 2006;32:1369–1375. Rix A, Palmowski M, Gremse F, Palmowski K, Lederle W, Kiessling F, Bzyl J. Influence of repetitive contrast agent injections on functional and molecular ultrasound measurements. Ultrasound Med Biol 2014;40:2468–2475. Salwei RM, O’Brien RT, Matheson JS. Characterization of lymphomatous lymph nodes in dogs using contrast harmonic and power Doppler ultrasound. Vet Radiol Ultrasound 2005;46:411–416. Schwarz KQ, Bezante GP, Chen X, Phillips D, Schlief R. Hemodynamic effects of microbubble echo contrast. J Am Soc Echocardiogr 1996; 9:795–804. Seiler GS, Brown JC, Reetz JA, Taeymans O, Bucknoff M, Rossi F, Ohlerth S, Alder D, Rademacher N, Drost T, Pollard RE, Travetti O, Pey P, Saunders JH, Shanaman MM, Oliveira CR, O’Brien RT, Gaschen L. Safety of contrast-enhanced ultrasonography in dogs and cats: 488 cases (2002–2011). J Am Vet Med Assoc 2013;242:1255–1259. Skrok J. Markedly increased signal enhancement after the second injection of SonoVue compared to the first - A quatitative normal volunteer study. 12th European Symposium on Ultrasound Contrast Imaging. Rotterdam, The Netherlands, 2007. Szebeni J. Complement mediated hypersensitivity to nanomedicines. Mol Immunol 2011;48:1715. Tang MX, Mulvana H, Gauthier T, Lim AKP, Cosgrove DO, Eckersley RJ, Stride E. Quantitative contrast-enhanced ultrasound imaging: A review of sources of variability. Interface Focus 2011; 1:520–539. Weskott HP. Emerging roles for contrast-enhanced ultrasound. Clin Hemorheol Micro 2008;40:51–71. Williams AR, Wiggins RC, Wharram BL, Goyal M, Dou C, Johnson KJ, Miller DL. Nephron injury induced by diagnostic ultrasound imaging at high mechanical index with gas body contrast agent. Ultrasound Med Biol 2007;33:1336–1344. Yanagisawa K, Moriyasu F, Miyahara T, Yuki M, Iijima H. Phagocytosis of ultrasound contrast agent microbubbles by Kupffer cells. Ultrasound Med Biol 2007;33:318–325.
http://dx.doi.org/10.1016/j.ultrasmedbio.2016.09.013
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Original Contribution QUANTITATIVE DIFFERENCES BETWEEN THE FIRST AND SECOND INJECTION OF CONTRAST AGENT IN CONTRAST-ENHANCED ULTRASONOGRAPHY OF FELINE KIDNEYS AND SPLEEN EMMELIE STOCK,* KATRIEN VANDERPERREN,* HENDRIK HAERS,* LUC DUCHATEAU,y MYRIAM HESTA,z and JIMMY H. SAUNDERS* * Department of Veterinary Medical Imaging and Small Animal Orthopedics, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium; y Department of Comparative Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium; and z Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium (Received 31 May 2016; revised 8 September 2016; in final form 9 September 2016)
Abstract—Contrast-enhanced ultrasound is a valuable and safe technique for the evaluation of organ perfusion. Repeated injections of ultrasound contrast agent are often administered during the same imaging session. However, it remains unclear if quantitative differences are present between the consecutive microbubble injections. Therefore, the first and second injection of contrast agent for the left renal cortex, renal medulla and the splenic parenchyma in healthy cats were compared. A lower peak intensity and area under the curve were observed for the first injection of contrast agent in the feline kidney, both for the renal cortex and medulla, and spleen. Moreover, for the renal cortex, the time-intensity curve was steeper after the second injection. Findings from the present study demonstrate that a second injection of contrast agent provides stronger enhancement. The exact mechanism behind our findings remains unclear; however, saturation of the lung macrophages is believed to play an important role. (E-mail: [email protected]) Ó 2016 World Federation for Ultrasound in Medicine & Biology. Key Words: Contrast-enhanced ultrasound, Microbubbles, Multiple injections, Kidney, Spleen, Cat.
diffuse renal disorders, both kidneys must be imaged. Additionally, patient motion during the study may induce the need for multiple injections of contrast agent. Some authors already mentioned that tissue enhancement after the first injection of microbubbles is subjectively less bright in comparison with subsequent contrast agent injections (Haers et al. 2013; Pey et al. 2011; Salwei et al. 2005). A study on contrast-enhanced ultrasound of liver and aorta in human patients confirmed this finding quantitatively (Skrok, 2007). In contrast, no significant differences were found between the first and second injection of contrast agent in mice liver and tumor tissue. However, a tendency toward a higher peak intensity (PI) after consecutive microbubble injections was noted (Rix et al. 2014). Thus far, it remains unclear if there are significant differences between the first and second injection of contrast agent. Nevertheless, it is of major importance to know if differences are present between both injections, as this could influence study results. Therefore, the purpose of this study was to compare several perfusion parameters between the first and second injection in the left kidney and spleen in healthy cats. The
INTRODUCTION Contrast-enhanced ultrasonography allows real-time visualization of macro- and microvascularization. Creating time-intensity curves and calculating several perfusion parameters allows quantification of tissue perfusion. The technique is extremely safe and can be performed in patients with renal impairment (Piscaglia et al. 2006; Seiler et al. 2013). Contrast-enhanced ultrasonography shows great potential for evaluation and diagnosis of various renal disorders in human and veterinary medicine (Granata et al. 2015; Haers et al. 2013). Repeated injections of contrast agent are most often required in contrast-enhanced ultrasound studies for different reasons. The first injection is sometimes used to optimize image settings. Multiple lesions may also be present, necessitating several injections of contrast agent to allow study of all lesions. In the assessment of Address correspondence to: Emmelie Stock, Department of Veterinary Medical Imaging and Small Animal Orthopedics, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium. E-mail: [email protected] 1
2
Ultrasound in Medicine and Biology
hypothesis is that the second injection of contrast agent will result in an increased enhancement of both the left kidney and the spleen; this will mainly be reflected by an increased PI and area under the curve (AUC). MATERIALS AND METHODS The experiments where approved by the Local Ethical Committee of Ghent University (EC2014/38). Seven healthy, purpose-bred laboratory cats with a mean age of 4.7 y and mean body weight of 3.1 kg were used. The cats were judged healthy based on physical examination, hematology, routine biochemistry, urinalysis and abdominal ultrasound. A 22-G catheter was placed in the cephalic vein. All cats were anesthetized with propofol (Propovet, Abbott Labs, Berkshire, UK), 6 mg/kg intravenous, maintained with additional boluses of 1 mg/kg on effect. A commercial contrast agent (Sonovue, Bracco, Milan, Italy) was administered at 0.15 mL per bolus, immediately followed by a 1-mL saline bolus. A three-way stopcock was used to avoid delay between the contrast agent and the saline flush. The same person performed the injections in a standardized manner. The cats received two consecutive injections, with approximately 10 min between the two injections. Between injections, the microbubbles were destroyed by setting the acoustic power at the highest value and scanning the caudal abdominal aorta, liver and spleen for approximately 2 min. A linear array transducer of 5–7.5 MHz on a dedicated ultrasound machine (MyLab 30CV, Esaote, Genoa, Italy), equipped with CnTI-contrast tuned imaging technology, was used. Machine settings were kept constant between the injections. A mechanical index of 0.09 was applied. The gain setting was adapted to a value just suppressing signals before arrival of contrast agent (55%– 58%). An image clip of 120 s at a frame rate 7.5 Hz was recorded for every injection. Imaging was started simultaneously with the contrast agent injection. The kidney was consistently imaged in a longitudinal plane. The spleen was imaged simultaneously with the left kidney. Quantitative analysis was performed using an offline image analysis program (Image J, US National Institutes of Health, Bethesda, MD, USA). The data were not linearized before quantification. Mean pixel intensity was measured every 1 s from 0 s to 70 s, every 2 s for 70 s to 90 s and every 4 s for 90 s to 120 s. A circular region of interest was drawn in the renal cortex and medulla at approximately the same location (ventral aspect of the renal cortex, central aspect of the renal medulla). The size of the region of interest was kept constant (1.2-cm diameter). A region of interest containing the complete visualized part of the spleen was drawn for the splenic pa-
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renchyma. Care was taken to avoid large vessels. The mean pixel intensity values were used to create timeintensity curves. The curves were analyzed for blood flow parameters representing blood volume (baseline intensity [BI], PI, AUC) and blood velocity (arrival time [AT], time-to-peak [TTP], wash-in/wash-out [Win/ Wout]). Wash-out was not calculated for the spleen, as only the first part of the wash-out occurred within the time the clip was captured (120 s). BI was defined as the intensity measured the first 3 s after contrast agent injection. AT was set at the point where the curve reached a value at least 5 mean pixel intensity values higher than BI. Wash-in was calculated from data points between 10% higher than BI and 90% of PI; wash-out from points between 90% of PI and T 5 120 s (end of the study). PI and AUC were corrected for the BI. Statistical test selection and analysis was performed by one of the authors (L.D.). The statistical analysis was based on a mixed model with the cat as the random effect and sequence (first or second injection) as the fixed effect, using F-tests at the 5% significance level (SAS Version 9.4, SAS Institute Inc, Cary, NC, USA). RESULTS AUC was significantly higher for the second injection compared to the first injection for both the renal cortex (p 5 0.01) and medulla (p 5 0.04). The PI was also higher for the second injection; this was only significant for the renal cortex (p 5 0.009) while a trend was present for the renal medulla (p 5 0.055). Additionally, for the renal cortex, the Win (p 5 0.01) and Wout (p 5 0.006) were less steep for the first injection compared to the second. No significant differences were present between the first and second injection for AT (cortex: p 5 0.4; medulla: p 5 0.3) and TTP (cortex: p 5 0.3; medulla: p 5 0.08). Similarly, the PI (p 5 0.007) and AUC (p 5 0.008) in the splenic parenchyma were significantly higher for the second injection compared to the first. The BI was consistently comparable for the first and second injection. Mean 6 standard error values of the renal blood flow parameters for the first and second injections are summarized in Table 1. Time-intensity curves comparing the first and second injection are represented by Figure 1. A contrast-enhanced ultrasound image of the left kidney and spleen of a cat is shown in Figure 2. DISCUSSION In the present study, significant differences were observed between the first and second injections of ultrasound contrast agent. The most reproducible differences between the two injections were a higher PI and AUC for the second injection, seen in the renal cortex, renal
Differences between the first and second injection of ultrasound contrast agent d E. STOCK et al.
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Table 1. Means and standard errors of perfusion parameters calculated from the renal cortex, renal medulla and splenic parenchyma in healthy cats, for the first and second injection of contrast agent Renal cortex Perfusion parameter BI PI AT TTP Win Wout AUC
Renal medulla
Spleen
First injection
Second injection
First injection
Second injection
First injection
Second injection
2.50 6 0.35 34.95 6 4.58* 7.51 6 0.61 41.75 6 2.36 1.17 6 0.19* 20.30 6 0,04* 2340.50 6 492.91*
2.96 6 0.35 51.13 6 4.58* 6.86 6 0.61 39.61 6 2.36 1.90 6 0.19* 20.43 6 0.04* 3769.82 6 492.91*
1.72 6 0.14 26.83 6 3.97 20.59 6 1.47 43.61 6 2.76 1.58 6 0.32 20.28 6 0.04 1077.03 6 322.53*
1.74 6 0.14 39.91 6 3.97 18.29 6 1.47 35.90 6 2.76 2.37 6 0.32 20.33 6 0.04 1983.53 6 322.53*
2.70 6 0.39 46.06 6 4.70* 7.22 6 0.55 61.03 6 1.29 1.74 6 0.45 3770.93 6 477.01*
3.13 6 0.39 59.60 6 4.70* 7.38 6 0.55 62.91 6 1.19 1.85 6 0.45 5235.02 6 477.01*
AT 5 arrival time; AUC 5 area under the curve; BI 5 baseline intensity; PI 5 peak intensity; TTP 5 time-to-peak; Win 5 wash-in; Wout 5 wash-out. * Value represents a significant (p , 0.05) effect.
medulla and splenic parenchyma. For the renal cortex, the wash-in and wash-out were also steeper after the second injection. In agreement with our results, a tendency toward a higher PI after consecutive microbubble injections was also noted in a recent study in mice tumors (Rix et al. 2014). However, in the study of Rix et al. (2014), the opposite effect, that is, a decreasing PI after consecutive injections, was observed in the liver. This might be due to short-term accumulation in liver Kupffer cells. In contrast, in a human study, an increased PI and AUC were noted in the liver after the second injection of microbubbles (Skrok, 2007).
Fig. 1. Mean time-intensity curves calculated from the renal cortex, renal medulla and spleen demonstrating a lower peak intensity and smaller area under the curve for the first injection of microbubbles. The time (in seconds) is displayed on the horizontal axis and the intensity (in arbitrary units [a.u.]) on the vertical axis.
There were no significant differences in BI between the first and second injection, indicating that no residual microbubbles were remaining in tissue of interest before the second injection. Between injections, the remaining microbubbles were destroyed by setting the acoustic power at the highest value and scanning the caudal abdominal aorta, liver and spleen for approximately 2 min. We avoided applying ultrasound waves of relatively high acoustic power on the kidney, as this could potentially cause capillary rupture (Miller et al. 2010; Williams et al. 2007). Rix et al. (2014) observed an increased heart rate after the first injection of microbubbles, which coincides with a faster circulation. This explains the faster washin. Moreover, an elevated heart rate and decreased TTP might result in a concentrated microbubble bolus, resulting in a higher PI. In humans, it was found that injections of relatively low volumes of phospholipid solutions might result in complement activation-related pseudo-allergy, causing a transient increase in heart rate and pulse pressure (Szebeni, 2011). The heart rate was not measured in our study. In another study by our research group, we did not observe a change in heart rate after consecutive contrast agent injections in healthy cats (unpublished results). Moreover, no effects on AT and TTP are present in the present study. It should be noted that the volume of the saline itself might cause an accelerated circulation in mice (Rix et al. 2014). In mice, short-term accumulation of microbubbles in the liver sinusoids and spleen might also explain the higher PI in tumors (Rix et al. 2014). It is unlikely that hepatic and splenic accumulation of contrast agent would explain the higher PI in the present study, as the majority of the microbubbles in the liver and spleen were destroyed by scanning these organs using high acoustic power. Moreover, it can be assumed that the low volume of contrast agent does not result in liver saturation in cats. In humans, injection of 1.2–1.4 mL microbubble solution does not result in liver saturation (Weskott, 2008). Another explanation for the stronger enhancement after the second injection might be saturation of the
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Fig. 2. Contrast-enhanced ultrasound images of the spleen and left kidney of a cat, illustrating a lower enhancement of after the first injection of contrast agent (left) compared to the second injection (right). Both images are obtained 30 s after contrast injection.
lung macrophages by the microbubbles (Skrok, 2007). This would allow more microbubbles to pass the lung and finally reach the systemic circulation. In pigs, administration of microbubbles resulted in an acute, transient and dose-dependent right ventricular dilation, increased pulmonary pressure and increased fractional shortening (Bramos et al. 2008). This could be caused by transient hypoxic pulmonary vasoconstriction. Pigs are known to develop severe pulmonary hypertension, which is likely to be caused by activation of pulmonary macrophages. Also, in dogs, increased pulmonary vascular resistance and pulmonary artery pressure was seen after administration of high doses of Levovist (Schwarz et al. 1996). No significant hemodynamic changes were observed at clinical, weight-adjusted doses in these dogs. The exact etiology of the effects on the pulmonary vascularization in these dogs remains unclear; transient pulmonary vasoconstriction might be a possible explanation (Schwarz et al. 1996). The last explanation that may be raised is a local effect caused by a vascular mediator. An interaction could be present between the contrast agent and the endothelium; moreover, the influence of the ultrasound waves on microbubbles could reinforce this effect. The combination of both could cause local activation of vasodilative substances. Wellperfused organs such as the kidneys and neoplastic tissues might therefore be more sensitive to this effect. Several factors may influence the presence and extent of differences between the first and second injection of contrast agent. First, the type of contrast agent may be an important factor. It is known that great differences are present in the extent and duration of phagocytosis by the reticuloendothelial system depending on the type of contrast agent (Tang et al. 2011). Generally, SonoVue shows a low uptake by Kupffer cells in contrast to other types of microbubbles, as do Sonazoid, Optison and Levovist (Yanagisawa et al. 2007). Moreover, species
may play an import role. As previously mentioned, pigs are prone to develop extensive pulmonary hypertension after activation of lung macrophages. To our knowledge, no information is available on this phenomenon in cats. Finally, the organ of interest may influence the results, as suggested in the murine study of Rix et al. (2014), which showed contrasting results in tumor tissue compared to the liver. In the latter study, peak enhancement in tumor tissue tended to increase after repeated microbubble injections, whereas the peak enhancement in the liver decreased steadily. Our study has some limitations. Cardiovascular parameters were not measured regularly throughout the study. However, data of other studies performed by our research group in feline patients revealed no significant changes in heart rate, blood pressure or respiratory rate after the first injection of contrast agent. The assessment of heart rate, blood pressure and even more interestingly, pulmonary artery pressure and the pressures in the different cardiac chambers, would deliver important information to distinguish between the possible causes for the low enhancement after the first injection. Body temperature was also not measured. It is known that hypothermia alters the blood flow and ultimately the distribution of contrast agent (Hyvelin et al. 2013). However, it is very unlikely that the cats would develop hypothermia, since the study duration was very short (15–20 min) and they were only superficially anesthetized. Moreover, only the first and second injections of contrast agent were studied. It would be interesting to have information about the subsequent injections of contrast agent. It remains unknown if differences are present between the second injection and following injections of contrast agent. Finally, we do not know how long this effect lasts. In other words, it is unclear if the same findings would be present if a second imaging session were performed in the same animal several hours later.
Differences between the first and second injection of ultrasound contrast agent d E. STOCK et al.
CONCLUSIONS We demonstrated clear differences are present between the first and second injection of contrast agent of the left kidney and spleen in cats. Less bright enhancement is achieved by the first injection. Unfortunately, we were not able to distinguish between the possible explanations for these findings. Activation/saturation of the lung macrophages is the most plausible explanation. Further research is necessary to clarify the mechanism of our findings and investigate if similar phenomena are present in other species and different organs. Our findings have important implications: the first injection should not be compared quantitatively to the second injection; additionally, use of a second injection is recommended for further analysis as it provides better enhancement. Acknowledgments—This research was supported by the Special Research Fund of Ghent University, Belgium (BOF grant 2015003301).
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