Background
In view of the debate on the safety of using ultrasound associated with microbubbles, we investigated the microvascular effects in situations of potential clinical interest.
Methods
Ultrasound, microbubbles, and their association were evaluated on the cheek pouch microcirculation in 96 male hamsters (n = 6 in each group): control; ischemia–reperfusion, 30 minutes total ischemia followed by reperfusion; hyperinsulinemia and hyperglycemia (substitution of the drinking water by 10% fructose solution for 3 months); and endotoxic shock (induced by Escherichia coli lipopolysaccharide). The heart rate, mean arterial pressure, number of rolling and adhered leukocytes, and arteriolar and venular diameters were analyzed.
Results
Heart rate and mean arterial pressure were lower in the endotoxic shock group than in the control group. Ultrasound alone or associated with microbubbles decreased the number of rolling and adhered leukocytes in the ischemia–reperfusion and hyperinsulinemia and hyperglycemia groups and increased venular diameter in the ischemia–reperfusion group.
Conclusion
The use of ultrasound decreases inflammation. Although it has not been established that pseudo-anaphylaxis can be assessed by intravital microscopy, microbubble administration does not increase venular leukocyte adhesion.
Ultrasound-enhancing contrast agents are microbubbles with a mean diameter less than 8 μm that cross the narrowest pulmonary capillary in the body and can be used in association with ultrasound to improve the visualization of blood flow. Their use increases the generated echo by 40 dB, allowing analysis of arterioles, venules, and capillaries. Ultrasound-enhancing contrast agents are gas bubbles that contain usually air or fluorocarbon and a covering shell (denatured albumin, surfactants, or phospholipids). A second generation of encapsulated microbubble agents, such as Definity (Bristol-Myers Squibb Medical Imaging Inc, North Billerica, MA), has been shown to behave in terms of microvascular rheology similar to red blood cells, an important point to establish its safety.
Because the use of microbubbles as a contrast agent is under debate in terms of safety and possible interference with the microcirculation, we investigated their effects on microvascular diameters and leukocyte/endothelium interaction representing the inflammatory response in control conditions and some pathologic situations, such as ischemia–reperfusion (I/R), hyperglycemia with hyperinsulinemia, and endotoxemia.
Microvascular diameter may be considered an indication of regional blood flow and as such is responsible for tissue nutrition and survival and leukocyte/endothelium interaction occurring mainly in post-capillary venules as an important step in the inflammatory cascade. Activated leukocytes have three consecutive stages of interaction with the endothelium: rolling, adhesion (adhered to the venular wall), and migration.
Pathologic conditions were chosen because of their prevalence in clinical practice. The number of persons with type 2 diabetes worldwide may increase from 135 million to more than 300 million by 2025 according to a recent estimation. The incidence of sepsis (10th leading cause of death in the United States ) is 57 per 1000 patients in Brazil and 75,000 cases per year in Germany. I/R mainly derived from coronary artery disease is obviously of interest. To the best of our knowledge, there are no studies evaluating the influence of microbubbles and ultrasound on the microcirculation in these conditions, although it is unknown whether pseudo-anaphylaxis can be detected with intravital microscopy. Therefore, the aims of the present study were to investigate the effects of the ultrasound contrast agent Definity combined or not to ultrasound on the microcirculation under the following conditions: 1) hyperinsulinemia and hyperglycemia (Hyp); 2) endotoxic shock (ES) induced by a bolus injection of Escherichia coli lipopolysaccharide; and 3) I/R using the hamster cheek pouch as an experimental model.
Materials and Methods
Animal Preparation
The study protocol was approved by the Ethical Committee of the State University of Rio de Janeiro, RJ, Brazil (CEA/215/2007). Ninety-six male Syrian golden hamsters ( Mesocricetus auratus, Botucatu, São Paulo, SP, Brazil), 7 to 15 weeks old, body weight between 100 and 140 g, were randomly divided into four experimental groups: control (C); I/R (30 minutes of total ischemia followed by reperfusion); Hyp, mimicking type 2 diabetes mellitus, obtained by substituting the drinking water by 10% fructose solution during 3 months; and ES, induced by a bolus injection of lipopolysaccharide from E. coli . Each experimental group had four subgroups according to the presence or absence of ultrasound and microbubbles: 1) without ultrasound and without microbubbles; 2) without ultrasound and with microbubbles; 3) with ultrasound and without microbubbles; and 4) with ultrasound and microbubbles. Animals received an appropriated laboratory diet (Nuvital; Nuvilab, Curitiba, PR, Brazil) and water ad libitum.
On the day of the experiment, anesthesia was induced by an intraperitoneal injection of sodium pentobarbital (0.2 mL/100 g body weight, Pentobarbital sodique, Sanofi, Paris, France, 60 mg/mL). After cannulation of the femoral vein, anesthesia was maintained with a bolus injection of alpha-chloralose (2.5 mL/kg body weight, Merck, Darmstadt, Germany). The cannula in the femoral vein was also used to inject microbubbles and lipopolysaccharide in appropriate groups. The femoral artery was also cannulated for blood pressure and heart rate measurements. Throughout surgery and subsequent experiment, the temperature of the animal was kept at 37.5°C with a heating pad controlled by a rectal thermistor (LTB 750 Thermostat System, Uppsala, Sweden). Tracheostomy was performed to facilitate spontaneous breathing. The hamster cheek pouch preparation was done according to procedures previously described by Duling and Svensjo and co-workers, modified by Bouskela and Grampp. The cheek pouch was prepared under a binocular stereomicroscope (Leitz, Wetzlar, Germany) and placed on the experimental chamber where it was gently everted. Large arterioles and venules were located and fixed with four needles into a circular well filled with silicone rubber to provide a flat bottom layer to avoid stretching or shrinking of the tissue. The pouch (125-150 μm thickness) was submerged in the superfusate HEPES-supported HCO 3 -buffered saline solution (composition in millimolar: NaCl 110.0, KCl 4.7, CaCl 2 2.0, MgSO 4 1.2, NaHCO 3 18.0, HEPES 15.39, and HEPES Na + salt 14.61) that continuously flushed the pool of the microscope stage at a rate of 4 mL/min whose temperature was maintained at 36.5°C. The pH was set to 7.40 by bubbling the solution continuously with 5% CO 2 and 95% N 2 . An incision was made in the upper layer, and a triangular flap was displaced to one side to produce a single-layer preparation. Magnification of 10 to 16× was used to remove fibrous, almost avascular, connective tissue covering the vessels with ophthalmic instruments. Cheek pouches with petechial hemorrhages or without blood flow in all large arterioles and venules were discarded. An intravital microscope (Leica DMLFS, Wetzlar, Germany, optical magnification 480×) coupled to a closed-circuit TV system was used, and preparations were allowed to rest for 30 minutes before measurements were taken.
Experimental Protocols, Microbubbles, and Ultrasound
Hyperglycemia with hyperinsulinemia was obtained by substitution of the drinking water by 10% fructose solution for 3 months; ES was obtained by an intravenous bolus injection of 5 mg/kg body weight of lipopolysaccharide from E. coli serotype 026:B6 (Sigma Chemicals Co, St Louis, MO) 24 hours before the experiment; and local ischemia of the cheek pouch was produced during 30 minutes with a cuff made of thin latex tubing that was mounted around the neck of the everted pouch where it leaves the mouth of the hamster. This cuff does not interfere with local blood flow, and an intracuff pressure of 200 to 220 mm Hg created by air compression with a syringe resulted in complete arrest of microvascular blood flow within a few seconds. Observations were made at 15 and 45 minutes after the onset of reperfusion.
For infusion, 0.1 mL of Definity microbubbles (Bristol-Myers Squibb Medical Imaging Inc, North Billerica, MA), mean size 1.1 to 3.3 μm, was diluted into 2.0 mL saline, agitated for 45 seconds, and infused with a pump (CMA/100 microinjection pump, Carnegie Medicine, AB, Sweden) at a continuous rate for a total of 1.05 mL during one hour. The infusion ended at the same time as the ischemia.
A portable ultrasound device, SonoHeart Elite (SonoSite Inc, Bothell, WA) was used to apply ultrasound (7.0 MHz, 1.2 mechanical index, 2.0 mPa peak pressure) to the hamster cheek pouch starting 30 minutes before the onset of the experimental period and ending after the ischemia. A convex transducer, appropriate to small animals, was used. Figure 1 shows the three protocols and respective time points of Definity infusion and ultrasound application. Infusion of isotonic saline solution, the same volume used for microbubbles, was made in control groups, without Definity, or ultrasound application.
Parameters Evaluated
The mean arteriolar and venular diameters, and number of adhered and rolling leukocytes were studied. In each preparation, three fields with one arteriole and one venule each were selected taking into account the possibility to return exactly to the same site (presence of fat cells, bifurcations) for consecutive measurements. Eighteen arterioles and eighteen venules were analyzed per group (3 arterioles/venules × 6 animals).
The mean value of microvascular diameters and leukocyte profile were calculated to decrease variability. Mean internal microvessel diameters were measured using an Image Shearing device, model 908 (Vista Electronics, San Diego, CA). Leukocytes were labeled in vivo by an intravenous injection of rhodamine-6G (Sigma Chemicals, St Louis MO) immediately before observations, initially 0.25 mL/100 g body weight in bolus and maintained with 10 μL/min until the end of measurement. Each field was recorded for 2 minutes with a videorecorder (Philips VR 999, Amazonas, Brazil) in sVHS format. Microcirculatory data were analyzed using the CapImage software.
Statistical Analysis
Results are shown as mean ± standard error of mean unless otherwise noted. For statistical analysis, Mann–Whitney U test and analysis of variance were used to compare groups and the comparison of the four groups at the same time was performed by Kruskall–Wallis and Dunn tests. P < .05 was considered statistically significant.
Results
Mean arterial pressure and heart rate were 101 ± 5 mm Hg and 422 ± 16 beats/min, respectively, at baseline. These parameters did not change significantly after I/R (90 ± 18 mm Hg and 412 ± 30 beats/min) and Hyp (93 ± 7 mm Hg and 381 ± 34 beats/min) but decreased in ES (59 ± 2 mm Hg and 365 ± 18 beats/min) ( Table 1 ).
| Control | Ischemia–reperfusion | Hyperglycemia and hyperinsulinemia | Endotoxic shock | |
|---|---|---|---|---|
| Mean arterial pressure (mm Hg) | 101 ± 5 | 90 ± 18 | 93 ± 7 | 59 ± 2 ∗ |
| Heart rate (beats/min) | 422 ± 16 | 412 ± 30 | 381 ± 34 | 365 ± 18 |
Inflammatory Profile
The mean number of rolling leukocytes was not affected by the presence of microbubbles in any studied group in the absence of ultrasound ( Figure 2 ). The comparison among the I/R, Hyp, and ES groups with controls at time points 0, 15, and 45 minutes showed no statistical differences.
The I/R, Hyp, and ES groups with or without microbubbles and without ultrasound showed an increased number of adhered leukocytes at time point 0 minutes (onset of reperfusion) in comparison with the control group. In addition, microbubbles did not influence the number of adhered leukocytes when injected without the application of ultrasound ( Figure 3 ). For each group, control, I/R, Hyp, and ES, there was no statistical difference among groups with and without microbubbles at each time point evaluated.
However, when ultrasound was applied without microbubbles there was a decrease in the number of rolling and adhered leukocytes in comparison with a similar group without microbubbles and without ultrasound for each time point evaluated on control, I/R, and Hyp groups ( Figures 2 and 3 ). The addition of microbubbles to ultrasound application elicited a further decrease in the number of rolling and adhered leukocytes on control, I/R, and Hyp groups.
Microvascular Diameters
The use of microbubbles or ultrasound did not interfere with the arteriolar diameter in any group. All diameters ranged between 18 and 34 μm. Therefore, the I/R, Hyp, and ES groups were not different from controls at the three studied time points ( Figure 4 ).
Venular diameters did not increase in the I/R, Hyp, and ES groups, compared with control, with or without microbubbles and without ultrasound. On the other hand, application of ultrasound by itself elicited venular dilation in the control and I/R groups ( Figure 5 ). The use of ultrasound combined to microbubbles increase venular diameters even further in the control and I/R groups.
Results
Mean arterial pressure and heart rate were 101 ± 5 mm Hg and 422 ± 16 beats/min, respectively, at baseline. These parameters did not change significantly after I/R (90 ± 18 mm Hg and 412 ± 30 beats/min) and Hyp (93 ± 7 mm Hg and 381 ± 34 beats/min) but decreased in ES (59 ± 2 mm Hg and 365 ± 18 beats/min) ( Table 1 ).
