Effect of MK-467 on organ blood flow parameters detected by contrast-enhanced ultrasound in dogs treated with dexmedetomidine

Authors

  • Flavia Restitutti,

    Corresponding author
    1. Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland
    • Correspondence: Flavia Restitutti, Department of Equine and Small Animal Medicine, P.O. Box 57 (Koetilantie 7), University of Helsinki, Helsinki 00014, Finland. E-mail: flavia.restitutti@helsinki.fi

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  • Merja R Laitinen,

    1. Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland
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  • Marja R Raekallio,

    1. Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland
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  • Mari Vainionpää,

    1. Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland
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  • Robert T O'Brien,

    1. Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Champaign, USA
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  • Erja Kuusela,

    1. Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland
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  • Outi M Vainio

    1. Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland
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Abstract

Objective

To evaluate the dexmedetomidine-induced reduction in organ blood flow with quantitative contrast-enhanced ultrasound (CEUS) method and to observe the influence of MK-467 on such reduction.

Study design

Randomized cross-over study.

Animals

Six adult purpose-bred laboratory beagle dogs (mean body weight 15.3 ± 1.9 kg).

Methods

Contrast-enhanced ultrasound was performed on six conscious healthy laboratory beagles. The animals on separate occasions underwent three treatments: awake without any medication (CTRL), dexmedetomidine 10 μg kg−1 (DEX) and DEX + MK-467 500 μg kg−1 (DMK) intravenously (IV). The kidney (10–15 minutes post-treatment), spleen (25–30 minutes post-treatment), small intestine (40–45 minutes post-treatment) and liver (50–55 minutes post-treatment) were examined with CEUS. A time curve was generated and the following perfusion parameters were analysed: arrival time (AT), time to peak from injection (TTPinj), peak intensity (PI) and wash-in rate (Wi). In addition to CEUS, renal glomerular filtration rate was indirectly estimated by the rate of iohexol elimination.

Results

AT and TTPinj were significantly higher for DEX than for CTRL in all studied organs. The same parameters were significantly higher for DEX than for DMK in the kidney, spleen and small intestine. PI was significantly lower for DEX than for CTRL or DMK in the kidney. Wi was significantly lower for DEX than for CTRL or DMK in the kidney and significantly lower than for CTRL only in the small intestine. Plasma concentration of iohexol was significantly higher after DEX than CTRL administration.

Conclusions

Contrast-enhanced ultrasound was effective in detecting DEX-induced changes in blood flow. MK-467 attenuated these changes.

Clinical relevance

Clinicians should consider the effects of the sedation protocol when performing CEUS. Addition of MK-467 might beneficially impact the haemodynamic function of sedation with alpha-2 adrenoceptor agonists.

Introduction

Alpha-2 adrenoceptor agonists, e.g. medetomidine and dexmedetomidine, are sedatives that exert their actions by stimulating central and peripheral alpha-2 adrenoceptors. Their sedative and analgesic effects are caused by the stimulation of central alpha-2 adrenoceptors, while the stimulation of peripheral alpha-2 adrenoceptors causes peripheral vasoconstriction (Clough & Hatton 1981; Horn et al. 1982), resulting in a transitory blood pressure elevation and bradycardia. During this hypertensive phase a higher peripheral vascular resistance occurs. After a single bolus, vascular resistance and blood pressure eventually return to near basal values (Pypendop & Verstegen 1998). Initially, bradycardia is attributed partly to a reflex elevation of vagal tone since baroreceptors are stimulated due to the rise in blood pressure (Doxey et al. 1981). After the hypertensive phase, bradycardia persists, but is the result of a reduction in sympathetic outflow due to the pre-synaptic effects of alpha 2 agonists, thus enhancing vagal tone (Sinclair 2003). Bradycardia and increased afterload result in decreased cardiac output (Thurmon et al. 1994; Sinclair et al. 2003), and consequently, a reduction in perfusion in the peripheral organs also occurs (Lawrence et al. 1996).

In order to avoid or attenuate the early hypertensive peripherally mediated phase that follows a bolus administration of an alpha2-adrenoceptor agonist, a peripheral alpha-2 adrenoceptor antagonist, MK-467 (also known as L659,066), has been studied in dogs and sheep. In both species, it has reduced the effects of dexmedetomidine (Pagel et al. 1998; Honkavaara et al. 2008, 2011; Raekallio et al. 2010) or medetomidine (Bryant et al. 1998; Enouri et al. 2008; Rolfe et al. 2012) on the cardiovascular system without compromising the sedation promoted by the central alpha-2 adrenoceptors (Honkavaara et al. 2008; Raekallio et al. 2010; Restitutti et al. 2011). However, the effects of the interaction of alpha-2 adrenoceptor agonists and MK-467 on blood flow in abdominal organs have not been studied to date.

Contrast-enhanced ultrasound (CEUS) is a new non-invasive approach to evaluate blood flow. It has been shown to be safe in dogs and cats (Nyman et al. 2005; Leinonen et al. 2011a; Seiler et al. 2013). In dogs, CEUS has been used to evaluate blood flow in kidneys (Wei et al. 2001; Waller et al. 2007), spleen (Ohlerth et al. 2007; Nakamura et al. 2009), liver (Ziegler et al. 2003; Nyman et al. 2005), jejunum (Jimenez et al. 2011), pancreas and duodenum (Johnson-Neitman et al. 2012). The method consists of the intravenous administration of an ultrasound contrast agent (USCA), enabling assessment of the tissue microcirculation in real time. The contrast agent, constituted of microbubbles, is a stable gas encapsulated by a shell of different composition. It remains exclusively in the intravascular space and is therefore considered a reliable imaging method for characterizing blood flow in tissue (Morel et al. 2000; Greis 2004). The microcirculation can be evaluated by CEUS qualitatively or quantitatively. Quantitative assessment provides more detailed information about the tissue blood flow, through the selection of a region of interest (ROI) and the analysis of certain blood flow parameters by generating a time-intensity curve (Li et al. 2006).

Iohexol is a non-ionic agent most usually used as a radiographic contrast medium. However, it is excreted unmetabolized in the urine by glomerular filtration only by the kidneys and does not undergo tubular secretion or re-absorbtion (Olsson et al. 1983). Thus, it is considered a validated marker to assess the glomerular filtration rate (Moe & Heiene 1995; Gleadhill & Michell 1996; Finco et al. 2001; Goy-Thollot et al. 2006a,b).

The primary aim of our study was to assess whether the dexmedetomidine-induced reduction in organ blood flow could be detected by CEUS. Our secondary aim was to observe the influence of MK-467 on organ blood flow parameters in dogs sedated with dexmedetomidine with the new, non-invasive technique.

Materials and methods

Animals

With the approval of the National Animal Experimentation Board, six adult purpose-bred laboratory beagle dogs aged three years, four castrated males and two spayed females, with a mean weight ± SD of 15.3 ± 1.9 kg, were used in this study. The animals were considered healthy according to clinical examination and blood tests. The dogs were housed in groups and fed with solid commercially available food twice daily. Food, but not water, was withheld 12 hours before the experiment.

Instrumentation

At the beginning of the trial, the animals were anaesthetized for catheter placement. Briefly, anaesthesia was induced with sevoflurane (SevoFlo; Abbott Laboratories Ltd., UK) in oxygen and delivered through a facial mask, the trachea was intubated, then the dogs placed in lateral recumbency. A 20G catheter (Optiva-2; Medex Medical Ltd., UK) was inserted percutaneously to the cephalic vein, and a 16G single lumen central venous catheter (CV-50016; Arrow International Inc., PA) was introduced through the jugular vein. Anaesthesia was then discontinued and the animals were allowed to recover for at least 30 minutes after extubation or until there were no signs of ataxia before the baseline measurements were obtained.

Study design

Each dog was given three treatments, and then imaged after each. The treatments were given on three separate days with a 14-day washout period, and administered in a Latin square, cross-over design, with a randomized order (Table 1) The three treatments were 1) conscious with no medication (CTRL), and after the following medication: 2) dexmedetomidine (Dexdomitor 0.5 mg mL−1, Orion Pharma, Finland) 10 μg kg−1 (DEX) and 3) DEX 10 μg kg−1 +MK-467 (Merck; Sharpe & Dohme, PA) 500 μg kg−1 (DMK). Both drugs were mixed in the same syringe and diluted with saline to a final standard volume of 10 mL and were administered in 30 seconds.

Table 1. Sequence of treatments underwent per dog after manual randomization following a latin square design
DogOrder of treatment
1DEXCTRLDMK
2DEXDMKCTRL
3DMKDEXCTRL
4CTRLDEXDMK
5CTRLDMKDEX
6DMKCTRLDEX

Additional to the imaging methods, iohexol concentration was measured to indicate differences in the glomerular filtration rate over a 2 hour period. Therefore immediately before treatment administration, iohexol (Omnipaque 300 mg mL−1; GE Healthcare, Finland) was injected IV at a dose of 1 mL kg−1 for later measurement of kidney function.

Organs were imaged according to the following sequence (Fig 1): kidney (10–15 minutes post-treatment), spleen (25–30 minutes post-treatment), small intestine (40–45 minutes post-treatment) and liver (50–55 minutes post-treatment). A concomitant study was performed during the sedation period (Restitutti et al. 2012).

Figure 1.

Timeline of the CEUS imaging organ sequence after awaken (CTRL) or treatment bolus (DEX, DMK) in 6 adult purposed-bread laboratory beagles.

Contrast-enhanced ultrasound imaging

Organs were imaged using a 2–5 MHz curvilinear transducer for the kidney and liver, and a 4–8 MHz linear transducer for the spleen and small intestine, with an ultrasound scanner (iU22 Ultrasound System; Philips Oy Healthcare, Finland). The hair was clipped over the ventral abdomen. Alcohol and gel were applied to the skin and the transducer positioned by hand by the same person (M.L.) during the imaging, who was unaware as to DMK and DEX but not to CTRL treatments. Care was taken to keep the transducer at the corresponding location and depth for each organ group. The mechanical index was maintained at 0.06–0.07 for the kidney, 0.05–0.06 for the spleen, 0.08 for the duodenum and 0.07–0.08 for the liver depending on the depth of view and the organ imaged. Standardized parameters included depth (kidney: 5.5–6 cm; spleen: 4 cm; duodenum: 3 cm; liver: 9 cm), time gain compensation and focal zones. One focal zone was placed at the level of or just below the organ imaged for each organ group separately. All dogs received at least two bolus injections (0.05 mL kg−1) of ultrasonic contrast medium (Sonovue, Bracco Imaging S.p.A., Italy), followed by a bolus of 5 mL of saline, for each imaged organ through a catheter in the cephalic vein. The injections were given in a standardized manner by the same person (F.R.) throughout the study. Four sets of 30 second video clips constituting in total 2 minutes of imaging series was recorded immediately after the completion of the contrast injection. Between the injections, an attempt was made to destroy the bubbles from the previous contrast injections with fundamental ultrasound at the level of the abdominal aorta or by imaging the heart. Time-intensity curves (TICs) were created online from the raw imaging data (QLAB – Region of Interest Quantification; Philips Oy Healthcare) of the first 30-second video clip. Measurements were made from the image sequence with the better quality according to the experience of the ultrasonographist, who was unaware of the identifier information on the ultrasound clips. Standardized TICs were obtained from selected regions of interest (ROI) in each organ and recording separately, representing the signal intensity (decibels) in relation to time (seconds). Care was taken not to include any adjacent tissues (mesentery, falciform fat) or larger vessels inside the ROIs. The functional perfusion parameters analysed were arrival time (AT [seconds]), defined as the time point when contrast level rises above baseline in the TIC, followed by a further rise (10% above baseline); time to peak intensity (TTPinj [seconds]), measured from the end of the injection, defined as the time elapsed to reach peak intensity following AT; peak intensity (PI [dB]), defined as maximal signal intensity at TTPinj, and wash-in rate (Wi [dB/s]), calculated from the data subset between 10% above baseline to 90% of PI. Data were tested for linearity by assessment of significance (< 0.05) and R-square (>0.7) to validate the use of linear regression analysis. In cases where the wash-in was very mild, which was often the case when estimating perfusion of the liver, linearity was not significant (> 0.05), the data were further cleaned from false pits and peaks and a smaller data subset was chosen for the estimation of wash-in rate; these are presented as WiNETcut. False peaks and pits were detected from the ultra sound image by observing when the ROI moved outside the target organ due to motion (e.g. respiratory movements), which lead to sharp pits caused by mesenteric fat or sharp peaks caused by larger vessels.

Plasma iohexol concentration analysis

Immediately before treatment administration (for DEX and DMK) or immediately before kidney imaging (for CTRL), 5 mL of blood was collected in EDTA tubes for baseline measurements of iohexol concentration. After the blood collection, 1 mL kg−1 of iohexol was injected IV. Two hours after iohexol administration, 5 mL of blood was collected and centrifuged, and the plasma was frozen at −20 °C for later iohexol analysis. In iohexol analysis, plasma samples were precipitated with 10% trifluoroacetic acid and analysed with an HPLC. The internal standard was 4-aminobenzoic acid (Pöytäkangas et al. 2010).

Statistical analysis

Normality of the data was tested using Shapiro–Wilk test. The differences between the two treatments and the controls were evaluated with an Analysis of Variance (anova) model, where treatment and the order of treatments given were included as explanatory factors. An anova model was constructed separately for different organs and CEUS parameters. Pairwise comparisons between the three groups were constructed using Tukey's Honestly Significant Differences (HSD) test. This test takes into account the multiplicity issues by adjusting the p-values of the comparisons. < 0.05 was considered significant.

Results

Figures 2-5 show the TIC obtained from the kidney, spleen, small intestine and liver, respectively, while the parameters derived from the TIC are summarized in Table 2. AT and TTPinj were significantly higher for DEX than for CTRL in all studied organs. The same parameters were significantly higher for DEX than for DMK in the kidney, spleen and small intestine (Table 2). PI was significantly lower for DEX than for CTRL or DMK in the kidney only (= 0.028 and = 0.011, respectively, Table 2). Wi was significantly lower for DEX than for CTRL or DMK in the kidney (= 0.036 and = 0.025, respectively, Table 2) and significantly lower than for CTRL only in the small intestine (= 0.009, Table 2). Plasma concentration of iohexol was significantly higher after DEX than CTRL administration (= 0.013).

Table 2. Contrast-enhanced ultrasound (CEUS) parameters and iohexol concentration in six untreated beagles (CTRL) and the beagles treated with dexmedetomidine 10 μg kg−1 IV (DEX) and DEX+ MK-467 500 μg kg−1 IV (DMK). Values are displayed as mean ± SD
 Time CEUS was performed*AT (seconds)TTPinj (seconds)Pi (dB)Wi (dB second−1)WiNETcut (dB second−1)WiNET (dB second−1)Iohexol (μg mL−1)
  1. *Time post IV treatment bolus; †Iohexol plasma concentration after 2 hours of IV administration of 1 mL kg−1 of iohexol 300 mg mL−1; ‡DEX different from CTRL (< 0.05); §DEX different from DMK (< 0.05). AT: arrival time; TTPinj: time to peak intensity; Pi: peak intensity; Wi: wash-in rate.

Kidney
CTRL10–15 minutes5.98 ± 3.319.56 ± 3.9535.47 ± 4.448.0 ± 3.0  305.33 ± 70.98
DMK4.24 ± 3.518.27 ± 4.7436.9 ± 5.438.4 ± 4.5  383.75 ± 57.35
DEX17.84 ± 6.09§25.63 ± 6.46§26.81 ± 6.48§2.8 ± 0.8§  429.03 ± 58.40
Spleen
CTRL25–30 minutes8.50 ± 6,8419.08 ± 6,5724.11 ± 7.621,1 ± 0,7   
DMK7.29 ± 3.0619.47 ± 5,8822.15 ± 2.230.7 ± 0.5   
DEX18.84 ± 2.66§29.04 ± 1.59§25.35 ± 1.921.1 ± 0.5   
Small Intestine
CTRL40–45 minutes7.47 ± 3.5411.62 ± 5.2734.82 ± 7.049.3 ± 5.7   
DMK10.07 ± 3.5614.49 ± 5.3229.17 ± 5.797.0 ± 2.9   
DEX16.53 ± 2.92§25.62 ± 5.08§25.81 ± 6.071.8 ± 0.5   
Liver
CTRL50–55 minutes9.43 ± 2.4216.33 ± 3.2727.80 ± 5.71 4.2 ± 3.03.4 ± 1.7 
DMK10.89 ± 4.5919.18 ± 6.8123.10 ± 3.35 2.8 ± 1.12.5 ± 0.8 
DEX18.73 ± 7.8027.60 ± 10.2822.99 ± 2.11 2.7 ± 1.31.9 ± 1.0 
Figure 2.

Mean generated time-intensity curve after contrast-enhanced ultrasound of the kidney cortex in six adult purpose-bred laboratory beagle dogs after treatment with dexmedetomidine 10 μg kg−1 (DEX – solid line) and DEX in combination with MK-467 500 μg kg−1 (DMK – dotted line) and while conscious after receiving no treatment (CTRL – dash line). No statistical analysis of the curve was performed.

Figure 3.

Mean generated time-intensity curve after contrast-enhanced ultrasound of the spleen in six adult purpose-bred laboratory beagle dogs after treatment with dexmedetomidine 10 μg kg−1 (DEX – solid line) and DEX in combination with MK-467 500 μg kg−1 (DMK – dotted line) and while conscious after receiving no treatment (CTRL – dash line). No statistical analysis of the curve was performed.

Figure 4.

Mean generated time-intensity curve after contrast-enhanced ultrasound of the small intestine in six adult purpose-bred laboratory beagle dogs after treatment with dexmedetomidine 10 μg kg−1 (DEX– solid line) and DEX in combination with MK-467 500 μg kg−1 (DMK – dotted line) and while conscious after receiving no treatment (CTRL – dash line). No statistical analysis of the curve was performed.

Figure 5.

Mean generated time-intensity curve after contrast-enhanced ultrasound of the liver in six adult purpose-bred laboratory beagle dogs after treatment with dexmedetomidine 10 μg kg−1 (DEX – solid line) and DEX in combination with MK-467 500 μg kg−1 (DMK – dotted line) and while conscious after receiving no treatment (CTRL – dash line). No statistical analysis of the curve was performed.

Discussion

Our findings indicate that DEX-induced changes in abdominal organ blood flow could be detected by CEUS, and the addition of MK-467 was effective in preventing those changes. However, the effect of time should also be taken into account when evaluating these results since DEX was administered as a single bolus and not as a constant-rate infusion, and each organ was measured at a separate time-point after drug administration. Comparisons in this study can thus be made only between treatments, not between organs. The ability of MK-467 to prevent the early effects of alpha-2 adrenoceptor agonists on the cardiovascular system of dogs has been demonstrated (Pagel et al. 1998; Enouri et al. 2008; Honkavaara et al. 2011; Rolfe et al. 2012), and the results of this paper support the ability of the antagonist to prevent decreases in blood flow of the abdominal organs. The alpha-2 antagonist atipamezole, by contrast, was not able to reverse the depressed blood flow in the heart and kidney of medetomidine-treated sheep (Talke et al. 2000).

In the kidney, the differences in CEUS parameters between DEX-treated dogs and dogs receiving the other two treatments (CTRL and DMK) were most obvious, which can be explained partially by the time of imaging and partially by the kidney's arterial vasculature, receiving marked blood flow from the abdominal aorta. In a previous study, the same dose of DEX as used in our study obtained its peak peripheral haemodynamic effects between 5 and 10 minutes post IV administration, when the cardiac index of treated dogs was approximately one-third of the baseline value (Honkavaara et al. 2011). In our study, the kidney was imaged at 10–15 minutes post-treatment, i.e. during the maximal effect. MK-467 was able to prevent the changes in renal blood flow caused by DEX. This observation is also supported by the iohexol plasma concentrations. Although glomerular filtration rate (GFR) was not calculated in this study, the differences in iohexol plasma concentrations between the treatments suggested that the decrease in the GFR after DEX was alleviated by MK-467, as GFR inversely correlates with iohexol plasma concentration (Gleadhill & Michell 1996; Goy-Thollot et al. 2006a). Thus, the addition of MK-467 could be beneficial for kidneys and GFR since this compound alleviates the effects of DEX on renal blood flow.

In the spleen, the addition of MK-467 alleviated the difference present between DEX and CTRL with AT and TTPinj, while no differences were found between any treatment with PI and Wi. AT and TTPinj detected in CTRL and DMK were comparable with those of a previous report (Ohlerth et al. 2007), but PI cannot be compared with earlier studies due to different settings of ultrasound equipment and different doses of USCA. In addition, the spleen may appear heterogeneous in the early phase of CEUS imaging (Catalano et al. 2005; Ohlerth et al. 2007), and anaesthesia has been reported to influence heterogeneity in cats (Leinonen et al. 2011b). We did not score the heterogeneity of the spleen in this study, and the effects of sedatives and anaesthetic drugs on early heterogeneity of the canine spleen remain to be determined.

Our study revealed that the addition of MK-467 also attenuates the DEX-induced reduction of blood flow in the small intestine. Cowles et al. (1999) have shown that transient reduction of blood flow does not interfere with motility of the ileum in dogs. The effects of transient reduced blood flow in the duodenum of dogs remain to be elucidated. Quantitative analysis of the small intestine has been carried out in cats (Leinonen et al. 2010; Diana et al. 2011) and dogs (Jimenez et al. 2011; Johnson-Neitman et al. 2012). However, comparisons between studies are hindered by differences in ultrasound machines, machine settings, contrast agents (products and dosing), anaesthesia and imaging protocols.

The liver was the only studied organ in which DMK was not statistically different from DEX, although the latter still differed from CTRL. A possible explanation for such results is the time of imaging. Being the last organ to be imaged (at 50–55 minutes post-treatment), the cardiovascular effects of DEX had already decreased (Honkavaara et al. 2011). Besides the time of imaging, this organ, with a dual blood flow (hepatic artery and portal vein), has several mechanisms to maintain its blood flow constancy, which must also be considered (Lautt 2007; Eipel et al. 2010).

Nyman et al. (2005) reported a faster time to peak enhancement of contrast agent in the liver of propofol-treated animals compared to awaken, conscious dogs. However, propofol affects the cardiocirculatory system (Langley & Heel 1988), increasing hepatic blood flow (Wouters et al. 1995; Zhu et al. 2008). The dose of propofol used in the study of Nyman et al. (2005) was not mentioned, thus no further comparisons with our results are possible.

In conclusion, changes in certain organ blood flow parameters, which were induced by DEX, could be detected with CEUS. Our results indicate that MK-467 attenuates the effects of DEX in organ blood flow. Furthermore, it should be noted that any sedation or anaesthesia protocol may have an effect on the blood flow parameters measured with CEUS. However, further studies are needed to overcome the varying temporal effects on the different organs to ascertain the full usefulness of this antagonist.

Acknowledgements

MK-467 was generously donated by Merck, Sharp & Dohme. The authors thank Vetcare Oy for financial support.