Sara Gargiulo, Adelaide Greco, Matteo Gramanzini, Silvia Esposito, Andrea Affuso, Arturo Brunetti, and Giancarlo Vesce
Sara Gargiulo, DVM/PhD, is a Research Fellow at the Bioimaging and Biostructures Institute, Italian National Council of Research, and at Ceinge Biotecnologie Avanzate scarl in Naples, Italy. Adelaide Greco, DVM/PhD, is an Adjunct Professor in the Biomorphological and Functional Sciences Department, University Federico II, and at Ceinge Biotecnologie Avanzate scarl. Matteo Gramanzini, DVM, is a Research Fellow at the Bioimaging and Biostructures Institute, Italian National Council of Research, and at Ceinge Biotecnologie Avanzate scarl in Naples, Italy. Silvia Esposito, DVM, is a Veterinarian at Ceinge Biotecnologie Avanzate scarl. Andrea Affuso, DVM, is a Veterinarian at Stazione Zoologica Anton Dohrn of Naples and IRGS-BIOGEM, Ariano Irpino, Italy. Arturo Brunetti, MD, is a Full Professor of Diagnostic Imaging, University Federico II, and is Group Leader at Ceinge Biotecnologie Avanzate scarl. Giancarlo Vesce, DVM, is a Full Professor of Veterinary Anesthesia at the School of Veterinary Medicine, University Federico II.
Address correspondence and reprint requests to Dr. Sara Gargiulo, Bioimaging and Biostructures Institute, Italian National Council of Research, Via T. de Amicis 95, 80145, Naples, Italy, or email sara.gargiulo@ibb.cnr.it.
Abstract
Animal experiments are necessary for a better understanding of diseases and for developing new therapeutic strategies. The mouse (Mus musculus) is currently the most popular laboratory animal in biomedical research. Mice imaging procedures are increasingly used in preclinical research because they allow in vivo monitoring and they are readily available for longitudinal and noninvasive studies as well as investigations into the evolution of diseases and the effects of new therapies. New imaging techniques and sophisticated laboratory animal imaging tools are currently producing a large body of evidence about the possible interference of anesthesia with different imaging methods that have the potential to compromise the results of in vivo studies. The purpose of this article is to review the existing literature on molecular imaging studies in mice, to describe the effects of different anesthetic protocols on their outcome, and to report our own experience with such studies.
Key Words: analgesia; anesthesia; chemical restraint; longitudinal studies; mice; preclinical research; small animal imaging
Introduction
Several techniques for structural and functional tomographic imaging in small rodents are utilized in the molecular imaging research field and include developing and testing novel molecular probes, characterizing morpho-functional animal models of human diseases, and monitoring novel therapeutic approaches. In our preclinical imaging laboratory, we conduct this research using the following state-of-the-art equipment: ultrasound biomicroscopy (UBM1), positron emission tomography (PET1) and single photon emission computed tomography (SPECT1), high-resolution x-ray computed tomography (micro-CT1), dual energy x- ray absorptiometry (DEXA1), laser doppler fluometry and optical imaging (bioluminescence and fluorescence).
Two fundamental principles must be taken into account in choosing a suitable anesthetic protocol for a specific imaging procedure. First, to guarantee a good imaging examination, personnel must completely immobilize and adequately numb the animal during the entire procedure to avoid movement artefacts. Second, for serial studies in which mice are examined repeatedly, researchers must select the proper anesthetic and patient care techniques. Investigators who study mice as models of human diseases and/or bear major pathologies (primarily strain-related metabolic deficiencies) must take into account the increased risk that is associated with anesthetic agents and techniques. They must first carefully assess the specific organ or function under study, and only then adopt an anesthetic strategy that avoids any interference of specific drugs with the outcome of the experiment and with the information provided by the imaging study. For example, ionizing radiation exposure or radionuclide or contrast media administration combined with anesthesia can adversely affect the health of the patient. In addition, although some imaging techniques (e.g., DEXA or bone CT studies) require minimal patient preparation, others (e.g., nuclear medicine techniques) entail more accurate, complex preliminary animal care and monitoring.
Anesthetic Protocols: Benefits and Risks
In the following sections, we describe examples of mice anesthetic protocols used to perform imaging procedures. Within the description, we identify in the limited allowable space in this article the beneficial and/or adverse effects of each protocol on the outcome of the patient and/or the study.
Ultrasound Biomicroscopy (UBM1)
The term ultrasound defines sound waves of frequencies that exceed the threshold of human hearing, which is 20 kHz. UBM is a high-frequency (20- to 100-MHz) pulse-echo ultrasound approach for imaging living biological tissues with a near-microscopic resolution. At these frequencies, it is possible to achieve a spatial resolution of 50 to 100 μm, linearly scaling with the beam frequency. UBM enables researchers to perform oncology studies (tumor growth or treatment effects) as well as developmental biology and cardiovascular (morphologic, functional, and hemodynamic) investigations, and to use microinjection techniques in target structures.
The advantage of UBM is that it allows real-time, noninvasive, and repeatable imaging of mice in the absence of ionizing radiation. Ultrasound examination affords the ultrasound specialist ample time to perform meticulous observation. Under these circumstances, inhalation anesthesia techniques are the first choice because they are safer and more uniform for long-lasting procedures than injectable anesthesia.
Echocardiography
Echocardiography is a well-known technique for analyzing transgenic and surgical models of cardiovascular diseases in mice. Because anesthesia has significant effects on cardiovascular performance, several studies have examined the influence of different anesthetic protocols on cardiac function by echocardiography. Some authors have reported a preference in conscious mice for echocardiography over the use of anesthetic agents (Esposito et al. 2000; Kiatchoosakun et al. 2001; Yang et al. 1999). However, measurements in awake mice require long conditioning sessions to train them to endure physical restraint to avoid autonomic responses such as bradycardia due to vagal stimulation, or tachycardia due to sympathetic stimulation. Yang and colleagues (1999) compared echocardiographic measurements in conscious and anesthetized mice, reporting a significant reduction of heart rate and cardiac contractility under pentobarbital or ketamine/xylazine anesthesia compared with values found in conscious mice, and assessing a mean heart rate of 658 beats/minute in control subjects. Similar heart rate and percent fractional shortening values were reported by Esposito and colleagues (2000) by echocardiography in conscious mice. Kiatchoosakun and colleagues (2001) reported heart rates of 612, 680 and 732 beats/minute, respectively, in conscious A/J, C57BL/6J, and FVB/N mice strains, but they concluded that they were unable to perform Doppler measurements in awake mice adequately despite training and manual restraint. Tan and colleagues (2003) also recommended performing echocardiograpy in conscious trained mice.
Thus, on the one hand, it appears that most authors view anesthesia as a valuable aid for providing sedation and easy restraint of mice, for reducing stress in the presence of sympathetic nervous system activation, and for enabling the acquisition of data during echocardiography. Yet on the other hand, anesthesia can significantly affect cardiovascular parameters and therefore the meaning of such measurements. Chari and colleagues (2001) compared the cardiovascular effects of the following two conventional anesthetic regimens used to assess cardiac structure and function: tribromoethanol (TBE1) 2.5% solution (12 μL/g [300 mg/kg]) and ketamine (65 mg/kg) mixed with xylazine (4 mg/kg) in Swiss Webster mice. They reported that the effects of these two anesthetic protocols on systolic and diastolic functions were related to their effect on heart rate. Specifically, ketamine-xylazine induced bradycardia, which at less than 300 beats/minute was consistent with a marked reduction of systolic function and with an increase of left ventricle end diastolic dimension (EDD1), whereas TBE allowed a higher and more steady heart rate (∼ 450 beats/minute). These authors reported no significant differences in derived load-dependent indexes of systolic function, concluding that both agents have myocardial depressant actions reflected by their effects on heart rate.
Roth and colleagues (2001) compared the effects of different anesthetic protocols: TBE 2.5% solution (12 μl/g [300mg/kg]), ketamine (50 mg/kg)/midazolam (3 mg/kg), ketamine (100 mg/kg)/xylazine (5mg/kg), and isoflurane (1.25% in oxygen) on echocardiographic measurements in C57BL/6J and C57BL/6N mice. They reported that major cardiac depression is produced by ketamine-xylazine and is consistent with bradycardia and reduced percent fractional shortening (% FS1) and enlarged EDD, whereas isoflurane yields major stability in % FS, a higher heart rate, and the greatest reproducibility in repeated studies. Under TBE anesthesia, the % FS was initially low but increased to values close to those found under isoflurane after 15 minutes of anesthesia. TBE also produced a more significant reduction of EDD in the 6J substrain of mice compared with the 6N substrain. The ketamine/midazolam combination produced similar effects on the cardiovascular system but with lower absolute values compared with those recorded under avertin anesthesia. That combination also produced a higher heart rate and % FS in 6J compared with 6N mice. The authors (Roth et al. 2001) concluded that the anesthetic protocol, timing of echocardiography, and genetic background all are critical variables during echocardiography in mice.
Takuma and colleagues (2001) compared the effects on cardiac function of 50 mg/kg of ketamine combined with either 6 mg/kg or 0.5 mg/kg of xylazine with those of isoflurane (0.6-2.2 %) in normal mice and in a surgically infarcted murine model. They concluded that heart rate and left ventricle systolic function values recorded during conscious studies closely resemble the physiological values both in control and infarcted mice, in contrast to the results obtained under ketamine/xylazine anesthesia. Isoflurane anesthesia also provided values closer to the physiological ones, but it caused a mild reduction of left ventricular systolic function compared with conscious animals.
Zuurbier and colleagues (2002) evaluated the effects of anesthesia on hemodynamic parameters of Swiss Webster, CD1, C57BL/6J, and Balb/c mice. The authors compared the effects of the intraperitoneal (IP1) injection of a fentanyl-fluanisone-midazolam combination with those of a ketamine-medetomidine-atropine combination and with those of isoflurane on mean arterial blood pressure and heart rate. They concluded that the choice between isoflurane and ketamine-medetomidine depends on the mouse strain used and on the parameter studied. In fact, ketamine-medetomidine resulted in higher mean arterial pressure for all four strains, whereas heart rate was higher under isoflurane anesthesia for all strains except Balb/c, which showed the same values under all anesthetic regimens.
Schaefer and colleagues (2005) investigated the influence of ketamine (100 mg/kg)-xylazine (1.25 mg/kg), of ketamine (100 mg/kg)–midazolam (3 mg/kg), and of TBE 2.5% solution (12 μL/g [300 mg/kg]) on diastolic left ventricular function in C57BL/6 mice. According to these authors, anesthesia with ketamine/xylazine is in combination with an initial 50% reduction of heart rate, but is characterized by more steady values in heart rate, % FS, and other diastolic function parameters. TBE and ketamine/midazolam instead induced a significant time-dependent increase in heart rate and a corresponding decrease of diastolic parameters. They concluded that to achieve consistent echocardiographic data in mice, investigators should use the same protocol and dose of anesthetic agents, and should observe the same interval from anesthetic induction to compare echocardiographic data.
Xu and colleagues (2007), searching for an optimal dosage, compared the effects of various ketamine-xylazine combinations on echocardiographic measurements in C57BL/6J mice. This mixture is largely used in echocardiographic studies due to its wide safety range, good sedation, and potential for reversing the α-2 agonist by yohimbine and atipamezole. They concluded that 100 mg/kg of ketamine, either alone or combined with 0.1 mg/kg of xylazine, produced an adequate 20-minute sedation for performing echocardiography that is characterized by a high and stable heart rate and has minimal effects on cardiac parameters. Ketamine and xylazine at 100 mg/kg and 10 mg/kg, respectively, produced a good immobilization but a significant alteration of basal cardiovascular parameters (lower % FS and heart rate).
Berry and colleagues (2009) also reported the use of light tranquillization, by midazolam alone (0.15 mg subcutaneously [SC1]), for performing echocardiography. Janssen and colleagues (2004) compared the systemic hemodynamic effects of four commonly used anesthetic regimens in mice: isoflurane, urethane, pentobarbital, and ketamine/xylazine. They concluded that isoflurane anesthesia preserves cardiac function better than other injectable anesthetics, and that the cardio-depressant effects of a ketamine-xylazine mixture are reversed in a few minutes by IP injection of atipamezole.
Reproduction and Perinatal Studies
Since the initial study of Turnbull and colleagues (1995) on in utero mice embryos, ultrasonography has been used increasingly both for imaging and staging early post-implantation embryos in utero, and for in utero microinjection of genes, viral vectors, and drugs. The issue of anesthesia is critical when performing foetal physiological studies, because embryos and fetuses are highly sensitive to anesthetics and to maternal homeostatic changes. Turnbull (2000), who anesthetized pregnant mice to perform in utero microinjections under pentobarbital or ketamine-xylazine anesthesia, discarded avertin because it causes arrhythmias of the embryonic hearts. Although in the past pentobarbital or ketamine and xylazine given by the IP route had been proposed in pregnant mice (Gui et al. 1996; Keller et al. 1996; Phoon et al. 2000; Woo et al. 1997), several authors have more recently advocated using isoflurane anesthesia in pregnant mice, both for transcutaneous echography and for performing transuterine microinjections after laparotomy (Junwu et al. 2008; Kulandavelu et al. 2003; Slevin et al. 2006). Isoflurane appears to be the anesthetic agent of choice for studies on cardiovascular function in embryos and fetuses despite the lack of specific data on mice (Mai et al. 2004). The advantages of inhalation anesthesia in pregnant mice include a reduced risk of intrauterine trauma in combination with injections, readily adjustable dosage, precise control of the duration of anesthesia, and rapid anesthetic induction and recovery.
Maintenance of appropriate maternal body temperature and oxygenation (Tobita et al. 2002) is critical for maintaining normal maternal and embryonic cardiovascular functions. Single or repeated exposure to anesthesia may induce developmental and/or long-term effects on embryonic and newborn mice, not yet studied in detail. It has been reported that chronic maternal exposure to light isoflurane anesthesia (4 hours/day daily from embryonic days 6-15) reduced embryonic growth and increased the incidence of cleft palate (Mazze et al. 1985); however, the effect of repeated exposure to deeper levels of anesthesia is largely unexplored. Zhou and colleagues (2002) observed minimal effects on the growth and cardiac diastolic function of newborn mice after repeated ultrasound examinations under isoflurane anesthesia. Analgesia after exposition of uterine horns is also a challenging aspect, because pain does reduce food and water intake after uterine surgery, which compromises maternal and fetal safety. Indeed, all anesthetics and central acting analgesics cross the placental barrier and are therefore contraindicated during pregnancy. Moreover, the co-administration of buprenorphine (2 mg/kg) with isoflurane has been reported to increase the mortality rate of mice (Janssen et al. 2004). In this case, the authors reported that “tender loving care” (including soft food, long drinking nipples, soft bedding, and a warm environment) was helpful as supportive therapy.
Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT)
PET and SPECT are nuclear medicine imaging techniques that produce a three-dimensional distribution of radiolabeled molecules or receptors of interest that are associated with body functional processes. These two small animal imaging tools emerged as a primary technique to investigate physiological as well as pathological processes at a molecular level in live animals. In the hybrid PET-CT scanners for small laboratory animals that are currently available, CT provides high-resolution anatomical information. SPECT and PET are also valuable in serial studies on the same animal for numerous research fields such as neuroscience, oncology, cardiology, pharmacokinetic studies of new drugs, and new radiopharmaceutical development for diagnostic and therapeutic use.
Mice PET/SPECT imaging techniques necessitate anesthesia for tracer injection and adequate immobilization to prevent movement during image scanning and to co-register nuclear medicine and CT or MRI images. Because PET and SPECT are functional techniques and anesthetic agents can affect metabolism and other physiological parameters, there is a growing interest in evaluating the impact of chemical restraint on tracer kinetics and biodistribution.
Fluorodeoxiglucose (18F-FDG)
Several studies have investigated the effects of fasting, warming, and anesthesia on small animal PET studies with FDG. The glucose analog 18F-FDG is a marker of cellular glucose consumption that is used extensively for studying heart, brain, and tumors. Toyama and colleagues (2004) compared the effects on 18F-FDG uptake in the heart and brain of nonfasted mice under ketamine/xylazine or isoflurane-anesthesia with those of conscious controls. The authors reported lower brain and heart 18F-FDG uptake in ketamine/xylazine-anesthetized mice compared with values in isoflurane-anesthetized mice and in conscious controls. Remarkably high uptake was observed in the hearts of isoflurane-anesthetized mice, while in the same group, brain 18F-FDG uptake was significantly lower compared with that of conscious controls.
In a similar study, we used an anesthetic protocol based on isoflurane induction, followed by the IP injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), for a 18F-FDG -PET examination of 37 mice following experimental myocardial ischemia at different intervals (24 or 48 hours; 1, 2, or 4 weeks) after surgical ligature of the left descending coronary artery. Atipamezole (1mg/kg) was administered IP at the end of each imaging procedure. This anesthetic protocol provided good immobilization for the entire duration of the PET-CT scan (~ 40 minutes) and allowed adequate visualization of 18F-FDG uptake in healthy myocardium as opposed to the lack of FDG uptake in the infarcted areas. We exercised care and used heating pads and infrared lamps to prevent hypothermia, and all of our animals survived the imaging procedure under this anesthetic protocol.
Lee and colleagues (2005) compared the effects of ketamine/xylazine and pentobarbital anesthesia in C57BL/6J mice with xenograft SC lung carcinoma neoplasia. They examined two groups of mice, which fasted respectively for 4 and 20 hours, and reported that after a 4-hour fast, both anesthetic protocols increased 18F-FDG concentration in the blood and reduced 18F-FDG uptake in myocardium and skeletal muscle without affecting tumor uptake. This finding can be explained by the significant increase in plasma glucose level brought about by xylazine and by the increased insulin level brought about by pentobarbital. After a 20-hour fast, plasma glucose levels were comparable between the awake and the anesthetized mice. The authors found that a reduced tumor:blood ratio enhanced the contrast between neoplastic and normal tissues.
Fueger and colleagues (2006) also investigated the impact of fasting and anesthesia on 18F-FDG biodistribution. They examined 18F-FDG uptake in severe combined immunodeficiency (SCID) mice with SC tumors that after an 8- to 12-hour fast, were kept warm under isoflurane or ketamine/xylazine anesthesia. They reported that warming significantly reduced the intense 18F-FDG uptake in brown adipose tissue and, like fasting, improved tumor visualization. Compared with ketamine/xylazine, isoflurane instead induced mild hyperglycemia, reduced 18F-FDG uptake by brown adipose tissue and muscle, and increased it in liver, myocardium, and kidney, because in small animals 18F-FDG uptake by the heart and lung can interfere with lung metastasis imaging.
Woo and colleagues (2008) investigated the impact of similar anesthetic regimens on 18F-FDG biodistribution in an effort to improve PET visualization of lung cancer metastases in C57BL/6J mice. They observed a dose-dependent increase in 18F-FDG uptake by the kidney, lung, intestine, and heart under isoflurane anesthesia brought about by a dose-dependent hypotension and reduced parenchymal blood flow proportional to anesthetic depth. Based on an increase in the intestinal 18F-FDG uptake in the mice anesthetized by ketamine (80 mg/kg) and xylazine (7 mg/kg), these authors concluded that the best anesthetic protocol for improving visualization of lung metastases in mice is isoflurane (0.5% in oxygen) during its biodistribution phase because it minimizes the heart and chest wall 18F-FDG uptake.
Radio-iodine meta-iodobenzylguanidine (MIBG1) is another tracer used for small animal imaging to monitor norepinephrine transport in neuroendocrine tumors or in myocardium. Ko and colleagues (2008) investigated the effects of the widely used anesthetic protocol ketamine xylazine on MIBG biodistribution in imprinting control region (ICR) mice. Whereas ketamine had the ability to inhibit norepinephrine reuptake in vitro, xylazine instead inhibited cellular MIBG uptake. In contrast to in vitro results, the authors showed that ketamine/xylazine enhanced the uptake of MIBG in myocardium, lung, liver, adrenal glands, and kidneys. Such behavior appears to be due to a reduced blood norepinephrine concentration and MIBG activity brought about by the inhibition of norepinephrine release via the α2-adrenoreceptors agonist. They concluded that ketamine/xylazine is an excellent method of anesthesia for MIBG imaging.
X-Ray Microcomputed Tomography (micro-CT)
The micro-CT technique uses x-rays to produce detailed three-dimensional images of soft and hard tissues. For soft tissue studies, the highest resolution is achieved by using a contrast agent and enhancing the x-ray attenuation of the investigated tissue. A high-resolution CT scanner (up to 30 μm) is a useful tool for preclinical studies on small laboratory animals in numerous research fields such as those involving investigations of bone pathologies (measurement of volumetric bone density and cortical bone parameters), tumors (visualization of primary neoplasia, metastasis, and angiogenesis), respiratory and cardiovascular pathologies, and phenotype characterization of transgenic mice models.
Mice imaged by micro-CT must be effectively immobile and must show fair muscle relaxation to achieve proper positioning. Unless the patient is deeply sedated, when the bed moves in the gantry, the vibrations and noise can stimulate mice and cause them to move. Body movements can create artifacts and quantification errors, especially in high-resolution modalities such as micro-CT and micro-MR (Chatziioannou 2002), and can thus interfere with co-registration and post-processing. The duration of a micro-CT acquisition can vary according to the resolution needed, to the extent of the imaging field (partial or total body), and to the need for obtaining a control followed by an enhanced (post-contrast) CT acquisition. Moreover, because CT is often performed immediately following a PET or SPECT examination to co-register images, the immobilization interval can extend from 10 minutes up to 1 hour. Ketamine/α-agonist combinations are suitable for this type of immobilization and muscle relaxation, and for reliable long-lasting effects (Deroose et al. 2007). Furthermore at the end of the procedure the α-agonist can be reversed by atipamezole to reduce recovery time. Nonetheless isoflurane inhalation anesthesia is the best choice particularly for long-duration examinations and when models of cardiovascular, hepatic or renal diseases are studied. Moreover, the use of a mechanical ventilator can be of additional help by allowing investigators to control the breathing rate and to reconstruct respiratory gated images. As an alternative, new specific “imaging anesthesia chambers” have recently been produced that provide constant anesthetic concentrations over long scanning periods. Such anesthetic chambers can be mounted on the micro-PET or micro-CT apparatuses, allowing reproducible positioning and easy co-registration of images (Bahadur et al. 2007; Haines et al. 2009; Suckow et al. 2008).
Dual Energy X-ray Absorptiometry (DEXA)
DEXA is a noninvasive imaging technique that allows serial measurements of body composition. The physical principle of this process is based on the differential attenuation of low and high energy x-rays between mineralized and soft tissues. Nonskeletal tissues are further assigned either to fat or to lean compartments. DEXA scans are often performed before and after treatments of different kinds including physical (e.g., exercise or whole body vibration), pharmacological, or surgical, to detect changes in body composition. This technique also is used to monitor the effects of genetic alterations, high-fat diets, or surgical ovariectomy on body composition (Brommage 2003; Hong et al. 2009; Nagy and Clair 2000).
DEXA also allows analysis of different skeletal parameters such as bone mineral density (g/cm2), bone mineral content (g), and bone area (cm2), and of soft tissue composition such as lean and fat mass (g). Additionally, the technique is useful for determining bone composition during growth as well as in adult subjects, and for evaluating specific body composition in, for example, obesity models. For these investigations, it is often necessary to anesthetize very young or fat mice, which can pose problems that are related to anesthetic conditions.
A total body DEXA acquisition lasts about 5 minutes and requires adequate immobilization and muscle relaxation to obtain a symmetrical body positioning from which the “region of interest” can be accurately drawn from total body images. Moreover, several subjects are often examined in the same DEXA session and monitored in serial studies, necessitating rapid anesthesia induction and recovery.
Ketamine-medetomidine (Cruz et al. 1998; Kiliç and Henke 2004) or ketamine-xylazine combinations proved to be very useful as a means of chemical restraint in our DEXA research protocols. Such restraint protocols provide good immobilization and allow personnel to awaken the animals quickly. In fact, in our experience mice start exploring the cage and eating shortly after the α2 agonist reversal. The residual effects of ketamine (tail rigidity, tail flicking, ataxia, hyperactivity) last up to 2 hours, or proportionally to the dose administered. We successfully adopted an anesthetic protocol with 40 mg/kg of ketamine and 0.8 mg/kg of medetomidine IP in 4 to 8 weeks with young, 22-g females , and a regimen of 75 mg/kg of ketamine/1 mg/kg of medetomidine and 50 mg/kg of ketamine/1 mg of medetomidine or 10 mg/kg of xylazine, respectively, in adult, obese female and male mice. Under these circumstances, isoflurane induction reduces the restraint stress and decreases the injectable drugs dosages.
Laser Doppler Flowmetry (LDF)
LDF is an imaging technique that enables investigators to measure local microcirculatory blood perfusion including capillaries, arterioles, venules, and shunting vessels. The technique is based on the “Doppler effect” in which the laser Doppler sends a monochromatic low-power laser beam toward the target tissue, collecting the radiations reflected by static structures and moving tissue particulates. The change in wavelength of the reflected radiation is a function of the relative velocity of the targeted object. Color-coded images of the blood perfusion in the microvasculature are thus created. Measurements are expressed as arbitrary perfusion units, which are used to calibrate the laser Doppler scanner. Scanning can be performed with semi-invasive laser Doppler probes and introduced inside body cavities (e.g., to measure mucosal perfusion of trachea, stomach, small intestine, and colon) or by a noninvasive scanner, which allows the study of the microcirculation over larger areas. LDF is used for monitoring superficial microcirculation activity in different research fields such as angiogenesis studies in ischemic limbs, treatment response to administration of vascular growth factors, or for the assessment of growth and neovascularization of skin tumors. Moreover, superficial cortical brain blood flow can be studied in stroke models after middle cerebral artery occlusion by noninvasively scanning through the mouse skull. In addition, gastrointestinal measurements can be performed, as an example, in small bowel transplantation surgery (Dindelegan et al. 2003).
LDF is a reliable and fast technique that provides information about peripheral blood perfusion and angiogenesis in mice. Scanning should be performed at a constant room temperature of about 25°C (77°F) after positioning the mice on a heating pad. It has been reported that recent shaving of animals or an uneven distribution of skin pigment of strains other than nude albino can induce bias in the measurements (Kragh et al. 2001). An anesthetic protocol that provides good immobilization and relaxation with short duration or quick reversibility should be used for LDF. Because the early peripheral vasoconstriction induced by α-agonists may interfere with study results, ketamine-α-agonist combinations are not suitable for LDF studies although ketamine-benzodiazepine combinations can be adequate for immobilization and do offer the opportunity to reverse one agent. Inhalation anesthesia is preferred because it offers the additional advantages of safe and fast anesthesia, complete recovery, and less interference with microcirculation.
Several preclinical studies have suggested using injectable anesthesia protocols to perform laser Doppler scanning in mice. Kragh and colleagues (2001) used LDF for noninvasive skin measurements of angiogenic and anti-angiogenic activity in nude mice anesthetized by ketamine (100 mg/kg) and xylazine (10 mg/kg), while Yang and colleagues (1999) used the same combination to evaluate skin and hind paw blood perfusion in a mouse model of atherosclerosis. Michauld and colleagues (2003) instead used the combination of ketamine (100 mg/kg)/ midazolam (5 mg/kg) to assess the blood perfusion in a murine ischemic hind limb model by laser Doppler. Bonheur and colleagues (2004) also used the technique to examine a murine surgical model of hindlimb ischemia-reperfusion injury under pentobarbital anesthesia. However, Baudelet and Gallez (2004) reported that pentobarbital (60 mg/kg), ketamine (80 mg/kg)/xylazine (8 mg/kg), fentanyl (0.078 mg/kg)/ droperidol (3.9 mg/kg), and isoflurane 1.5% all influenced hind limb blood flow in mice in different degrees. These authors observed a muscular blood flow reduction in ketamine/xylazine-anesthetized mice, and an even more marked reduction under pentobarbital anesthesia. Yet in fentanyl-droperidol- and isoflurane-anesthetized mice, they observed no differences in muscle perfusion. Furthermore, they reported similar drug-related effects on blood perfusion of tumors arising from the right gastrocnemius muscle. Thus, all of the studies described above indicate that investigators should carefully select an anesthetic protocol during the evaluation of blood perfusion, and should carefully consider the relevant effects of different anesthetics on regional blood flow.
LDF can be used in mice to measure cerebral blood flow. It is well known that anesthesia, like physiological sleep, affects cerebral blood flow and brain metabolic rate. For this reason, it is important to understand the effects of an anesthetic protocol on cerebral hemodynamics because they can interfere with the results of a study. Except for ketamine and tiletamine, which increase cerebral blood flow and intracranial pressure, all anesthetics decrease cerebral blood flow and intracranial pressure along with brain metabolic rate and activity. Using LDF, Okamoto and colleagues (1997) showed that in mice, as in other species, isoflurane increases regional cerebral blood flow in a dose-dependent manner. Also using LDF, Kehl and colleagues (2002) monitored the isoflurane-induced cerebral hyperemia in mice. Several studies in which cerebral blood flow in rats was measured by LDF or by other means reported that different anesthetic agents such as dexmedetomidine (Ganjoo et al. 1998) and the ketamine/xylazine combination (Lei et al. 2001) decrease cerebral blood flow effects that can be anticipated also in mice.
Optical Imaging
Optical Imaging is a highly sensitive technique that is based on the detection and quantification of light originating from luciferase-labeled cells or micro-organisms in living rodents. The technique has been applied in different research fields such as imaging of in vivo mice tumor cell metastasis and subsequent response to treatment as well as tumor cell proliferation studies and tumor protein interactions (Inoue et al. 2010; Klerk et al. 2007). Optical imaging is noninvasive and requires very short acquisition times (typical range of 1-180 seconds).
Optical imaging techniques are very useful in longitudinal studies on animal groups in that examinations can be completed in a few minutes. Investigators should therefore select an anesthetic protocol that provides good immobilization and muscle relaxation in mice, and that is either short-acting or quickly reversible. A ketamine-α2-agonist combination is most suitable. Most optical imaging scanners for small laboratory animals are also currently equipped with an inhalation anesthesia apparatus, which favors the technique. Under these circumstances, Cui and colleagues (2008) reported that the intensity of the bioluminescence signal can be affected by several factors such as patient positioning and anesthetic protocol. Using bioluminescence, they specifically examined athymic nude mice that had been inoculated with intracranial glioblastoma or SC melanoma expressing “firefly luciferase.” They compared the effects of three different anesthetics protocols: isoflurane 1.5%, ketamine (80 mg/kg)/xylazine (10 mg/kg), and avertin (240 mg/kg) on the intensity of the bioluminescence signal. These authors showed that different anesthetics significantly affect the photon count, and they reported the highest value under ketamine/xylazine and the lowest under isoflurane anesthesia, while the peak time of the bioluminescence signal was relatively independent from the anesthetic protocol. They also concluded that because positioning can have a profound impact on the consistency of signal measurements, it is important for improving the accuracy of serial studies to position the animals consistently.
Keyaerts and colleagues (2012) tested the effects of isoflurane, sevoflurane, desflurane, ketamine, xylazine, medetomidine, pentobarbital, and avertin as inhibitors of the luciferase enzyme reaction. The highest signal intensities were measured in pentobarbital anesthetized mice, followed by avertin, while isoflurane and ketamine/medetomidine anesthetized mice showed the lowest photon emission, They concluded that although strong inhibitory effects of anesthetics are present in vitro, their effect on in vivo BLI quantification is mainly due to their hemodynamic effects .
Magnetic Resonance Imaging (MRI)
The MRI technique consists of applying to tissues a strong magnetic field along with radio waves pulses to induce the physical phenomenon of “resonance” in their hydrogen nuclei and, subsequently, to detect and quantify the radio waves (the “signal”) that such proton “relaxation” generates. MRI provides morphological information on brain, heart, and body structures, on tissue chemical compositions, and on physiological phenomena such as blood flow. MRI provides a better soft tissue contrast than CT, which is enhanced by using “paramagnetic contrast agents.” Nevertheless, problems related to anesthesia in MRI include the following: (1) invariable presence of a strong magnetic field and radio frequency pulses that interfere with ferromagnetic components of anesthesia and monitoring devices; (2) heavy noise during image acquisition, which can stimulate motion in animals; and (3) considerable length of scanning time.
The use of inhalation anesthesia for MRI studies requires specially designed ventilators and vaporizers devoid of ferromagnetic parts. However, if an investigator has chosen an injectable anesthetic protocol, it should provide deep, long-lasting sedation and immobilization during MRI scanning to avoid movement artifacts. Indeed, inhalation anesthesia represents the first choice in MRI studies on mouse developmental biology for the same reasons discussed above for ultrasound biomicroscopy application in pregnant mice (Junwu et al. 2008; Kulandavelu et al. 2003; Slevin et al. 2006).
Kober and colleagues (2004) compared the assessment of cardiac performance by MRI in ketamine/xylazine or isoflurane-anesthetized mice, and they recommended the latter because it caused less depression of cardiac function compared with injectable anesthetics. They also recommended keeping isoflurane concentration at the lowest setting due to its respiratory depressant, dose-dependent effect. Recently, several studies by Cine MRI on genetically engineered models of cardiovascular disease in mice were reported under isoflurane anesthesia (Hove et al. 2005; Jia et al. 2005; Voros et al. 2006; Zhou et al. 2003).
Kober and colleagues (2004) also evaluated the influence of different anesthetic protocols on regional myocardial blood flow measurements in mice. They assessed the effects of isoflurane and those of ketamine (100 mg/kg) /xylazine (5 mg/kg) on coronary blood flow in C57BL/6J mice. They found that under ketamine/xylazine anesthesia, the heart rate was reduced roughly by 50%, whereas respiratory rate stayed close to physiological values. In addition, the raw image quality was not significantly altered by breath motion because chest movements were of smaller amplitude compared with isoflurane anesthesia. Myocardial blood flow under ketamine/xylazine was not significantly lower than under MAC 1 isoflurane (1.25 %), but a dramatic decrease was observed under 2% isoflurane anesthesia due to vasodilation and increased capillary flow.
Berry and colleagues (2009) evaluated the effects of deep sedation or general anesthesia on cardiac function both in normal and ischemic heart failure mice models. They compared the effects of 1% isoflurane with that of the morphine (4.5 mg/kg)/midazolam (9 mg/kg) combination, by SC injection, on heart rate and left ventricular ejection fraction. They reported that in healthy mice, deep sedation by morphine-midazolam causes significantly less depression of heart rate and ejection fraction compared with isoflurane, while in heart failure mice, there was a nonsignificant trend toward lower heart rates under isoflurane anesthesia compared with the injectable protocol. These authors conclude that deep sedation by morphine and midazolam causes less cardiac depression than isoflurane, and is most useful for MRI studies that last less than 20 minutes in allowing a comparable image quality.
Using BOLD-MRI (blood oxygen level dependent MRI), Baudelet and Gallez (2004) examined the evolution of tumor oxygen consumption and blood flow in a muscle tumor model of nuclear MRI mice anesthetized by pentobarbital (60 mg/kg), ketamine (80 mg/kg)/xylazine (8 mg/kg), fentanyl (0.078 mg/kg)/droperidol (3.9 mg/kg), or isoflurane 1.5%. According to these authors, the tumor signal intensity was dramatically decreased by the administration of all of the anesthetic protocols except for 1.5% isoflurane. In the studies that used isoflurane anesthesia, the measurements correlated well with the values of oxygen assessment by luminescence-based probes. They therefore recommended careful monitoring of the anesthetic effects in studies on new therapeutic approaches that alter tumor hemodynamics.
Conclusions
The selection of an anesthetic protocol for mice used in preclinical research with particular imaging techniques depends on a variety of factors as discussed above. Essentially any anesthetic choice, dosage, and regimen should be customized to the experimental population, the individual animal, and the experimental procedure. Indeed, animal evaluation, preparation, and anesthetic protocol can significantly influence both the quality and the experimental results of imaging studies.
The selection of the most suitable anesthetic protocol in laboratory mice should be adapted to strain, age and weight, model of the disease investigated, type and length of the procedure, and aim of the study. Just as in vivo imaging of small animals has emerged as an important tool in biomedical research, so also should animal care and anesthesia be adapted to the different types of imaging techniques. Additional studies are needed for a better refinement of animal handling and anesthesia, for optimizing the results of imaging techniques, and for simultaneously improving animal welfare.
1Abbreviations that appear ≥3x throughout this article: 18F-FGD, fluorodeoxyglucose; CT, computed tomography; DEXA, dual energy x-ray absorptiometry; EDD, end diastolic dimension; IP, intraperitoneal; LDF, laser Doppler fluometry; MIBG, meta-iodobenzylguanidine; MRI, magnetic resonance imaging; % FS, percent fractional shortening; PET, positron emission tomography; SC, subcutaneous ; SPECT, single photon emission computed tomography; TBE, tribromoethanol; UBM, ultrasound biomicroscopy.
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