Anesthetic Techniques Influence the Induction of Pulmonary Capillary Hemorrhage During Diagnostic Ultrasound Scanning in Rats

Pulsed ultrasound (US) can induce pulmonary capillary hemorrhage in mammals, which raises some concern for diagnostic US in medicine. This bioeffect was discovered more than 20 years ago1 and was evahiated in subsequent research for dependence on various pulsed US parameters to assess the possible risk for diagnostic US. Reported exposure thresholds for pulmonary capillary hemorrhage found in studies of the response to varied peak rarefactional pressure amplitude were reviewed by the American Institute of Ultrasound in Medicine.2 The results were interpreted in terms of the mechanical index(MI), which is an exposure index displayed on the screens of most US machines. The MI concept can be used for research by adjusting the peak rarefactional pressure amplitude measured in water for attenuation and dividing by the square root of the pulse center frequency. The thresholds, which were obtained mostly in anesthetized mice, corresponded roughly to an MI value of0.63.2 For reference, the guideline upper limit for diagnostic US is an MI of 1.9.3 Ultrasound-induced pulmonary capillary hemorrhage was found at about the same derated peak rarefactional pressure amplitudes in mice, rats, rabbits, pigs, and monkeys. A meta-analysis ofl4 studies in mice and rats found that the pulmonary capillary hemorrhage effect had widely varying thresholds and appeared to depend on physical parameters, including pulse duration, pulse repetition frequency, and exposure duration, in addition to peak rarefaction al pressure amplitude and frequency.4 In another authoritative reviewby theAmerican InstituteofUltrasound in Medicine, patient pulmonary US exposure was expected to occur during incidental exposure to echocardiography, which was not thought to pose a consistently defined risk.5 However, this overview of the pubnonarycapillary hemorrhage problem has changed in recent years for two reasons. First, most of the available data were obtained with pulsed US from laboratory exposure systems, and the applicability to actual diagnostic US was uncertain. Diagnostic US was used for studies in monkeys6and rats,7 but the results were mixed. Astudyofhuman hingsafterechocardiography indicated that 3.5-MHz diagnostic US up to 2.4 MPa (MI = 1.3),which was thoughtto interact with the lung as incidental exposure, did not cause pulmonary capillary hemorrhage.8 In a recent study in rats from our laboratory, diagnostic US from a 7.6-MHz linear array was used to directly scan the right lung ofanesthetized rats in a warmed water bath.9 The image displayed growing comet tail artifacts, which were indicative of pulmonary capillary hemorrhage on the surface of the lung. Pulmonary capillary hemorrhage was observed for several groups of rats scanned ata range of MI settingsfor 5 minutes, and a threshold for hemorrhage occurrence was indicated for an MI of about 0.44. This result appears to be lower than expected from the meta-analysis of physical parameters.4 The etiology of pulmonary capillary hemorrhage induced by diagnostic US remains poorly understood. Second, direct pulmonary US examination has become a valuable diagnostic aid for pneumonia, pulmonary edema and effiision, pubnonary embolism, atelectasis, diffuse parenchymal disease, adult and newborn respiratory distress syndrome, and lung cancer. 10 Diagnostic US imaging has largely replaced chest radiography for pleural effusion, pneumothorax, hemothorax, and posttraumatic lung contusion. 11– 12 Pubnonary US is rapidly becoming routine in intensivecare, 13-15 emergencycare, 16and otherpoint-ofcare settings. 17 This adoption of routine pulmonary US completely changes the diagnostic US-induced pulmonary capillary hemorrhage problem of incidental exposure to one of direct, deliberate examination. The problem requires further examination from newperspectives to clarify potential patient risks and provide guidance for the safe use of pulmonary US. One aspect of the problem in need of elaboration is the role ofbiological and physiologic factors. Age is one biological factor that has been explored. For mice and rats, pulmonary capillary hemorrhage thresholds appeared to have little dependence on age.18,19 Neonatal swine also had pulmonary capillary hemorrhage thresholds comparable to those ofadult mice.20 For older pigs, the experimentation is more difficult due to the size of the animals, but this animal model more closely simulates humans. O’Brien etal21found that older pigs were more sensitive than neonatal or middleaged pigs to 3.1-MHz pulsed US for 10-second exposures. Other physiologic conditions may also be important. One key factor for research is the use of anesthetics. Forgeneral anesthesia in mice and rats, intraperitoneal injection of ketamine plusxylazine is typically used; for example, studies of rats and mice reviewed by Church and O’Brien4 all used ketamine plusxylazine. The doses were 87 mg/kg ketamine plus 13 mg/kgxylazine in rats or mice,21-24 100 mg/kg ketamine plus 10 mg/kg xylazine in rats,7 and 200 mg/kg ketamine phis 10 mg/kgxylazine in mice. 1,17,25,26 The presently recommended anesthetic doses of 91 mg/kg ketamine and 9 mg/kgxylazine were used in our previous study.9 Ketamine is a dissociative anesthetic, which immobilizes with little respiratory depression, whereas xylazine is a sedative and analgesic, which produces substantial cardiovascular and respiratory depression.27 Xylazine has been reported to induce pulmonary edema in rats at relatively high dosesof 21 to 45 mg/kg.28,29 In a previous study using bronchoalveolar lavage albumin levels as representative of permeability injury, the normal doses of xylazine used for anesthesia did not produce a significant injury when compared with ketamine alone. However, the omission ofxylazine reduced the size of the comet tail artifacts produced during US scanning and significantly reduced the erythrocyte count in the bronchoalveolar lavage fluid found after scanning.30 Statistical analysis using analysis of variance (ANOVA) suggested that a synergistic interaction occurred between the USscanningand the use of xylazine. The goal of this study was to examine the influence of commonly used anesthetics and drug combinations on the induction of pulmonary capillary hemorrhage by diagnostic US scanning, using our previous methods.9 A common anesthetic for rodents is pentobarbital. This barbiturate yields prolonged anesthesia and can cause cardiovascular and respiratory system depression.27 Inhalation anesthesia is typically used for larger animals but is also valuable for short-term anesthesia in rodents. Isoflurane typically is applied at 1% to 5% in oxygen or air from a vaporizing machine. Thresholds were determined using ketamine withoutxylazine, pentobarbital, and isoflurane. Pubnonary capillary hemorrhage results for ketamine with and without xylazine were compared. In addition, the effect of the addition of xylazine to is of huane was tested. The results show substantial influences of the specific anesthetics on US-induced pulmonary capillary hemorrhage.