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Anesthesia and Other Considerations for in Vivo Imaging of Small Animals
Isabel J. Hildebrandt, Helen Su, Wolfgang A. Weber
Isabel J. Hildebrandt, PhD, is a research associate; Helen Su, PhD, is a postdoctoral fellow; and Wolfgang A. Weber, MD, is an associate professor of Molecular and Medical Pharmacology, all in the Department of Molecular and Medical Pharmacology at the David Geffen School of Medicine at the University of California, Los Angeles.
Address correspondence and reprint requests to Dr. Wolfgang A. Weber, UCLA School of Medicine, Nuclear Medicine, AR-264 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90095-6942 or email wweber@mednet.ucla.edu.
Abstract
The use of small animal imaging is increasing in biomedical research thanks to its ability to localize altered biochemical and physiological processes in the living animal and to follow these processes longitudinally and noninvasively. In contrast to human studies, however, imaging of small animals generally requires anesthesia, and anesthetic agents can have unintended effects on animal physiology that may confound the results of the imaging studies. In addition, repeated anesthesia, animal preparation for imaging, exposure to ionizing radiation, and the administration of contrast agents may affect the processes under study. We discuss this interplay of factors for small animal imaging in the context of four common imaging modalities for small animals: positron emission tomography (PET) and single photon emission computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), and optical imaging. We discuss animal preparation for imaging, including choice of animal strain and gender, the role of fasting and diet, and the circadian cycle. We review common anesthesias used in small animal imaging, such as pentobarbital, ketamine/xylazine, and isoflurane, and describe techniques for monitoring the respiration and circulation of anesthetized animals that are being imaged as well as developments for imaging conscious animals. We present current imaging literature exemplifying how anesthesia and animal handling can influence the biodistribution of PET tracers. Finally, we discuss how longitudinal imaging studies may affect animals due to repeated injections of radioactivity or other substrates and the general effect of stress on the animals. In conclusion, there are many animal handling issues to consider when designing an imaging experiment. Reproducible experimental conditions require clear, consistent reporting, in the study design and throughout the experiment, of the animal strain and gender, fasting, anesthesia, and how often individual animals were imaged.
Key Words: anesthesia; animal handling; fasting; imaging; mouse; PET; small animal
Introduction
Small animal imaging is used in studies of many different models of human diseases including cancer (Gambhir 2002), immunological responses (Hildebrandt and Gambhir 2004), cardiovascular disorders (Jaffer et al. 2006), neurodegenerative disorders (Jack et al. 2007), and transgene expression (Serganova and Blasberg 2005). A number of imaging modalities are available for small animal research, all with the common advantage of allowing serial studies with a single animal and the ability to localize altered biochemical and physiological processes. However, investigators planning an imaging experiment must consider three important differences among the imaging modalities in terms of animal handling: the need to inject an imaging probe, the sensitivity for detection of the imaging probe, and possible side effects from ionizing radiation or the injected compound.
In the relatively young field of small animal imaging, there are few published studies on the impact of animal handling, but they clearly indicate that animal handling can significantly influence the results of various imaging modalities. This review summarizes the reported findings and describes the issues to be addressed when considering the preparation of animals for imaging studies.
Imaging Modalities
Various techniques exist for small animal imaging, including position emission tomography (PET1), single photon emission computed tomography (SPECT1), computed tomography (CT1), magnetic resonance imaging (MRI1), and optical techniques such as bioluminescence and fluorescence imaging. Because a number of reports have reviewed these techniques (Gambhir 2002; Hildebrandt and Gambhir 2004; Jaffer et al. 2006; Levin 2005; Serganova and Blasberg 2005), in this article we focus on how animal preparation differs among these imaging modalities and how the imaging procedures themselves can affect animal physiology.
PET and SPECT
Positron emission tomography (PET) and single photon emission computed tomography (SPECT) rely on detection of photons emitted from radiolabeled probes in the body. PET records the coincident detection of high-energy gamma rays emitted from positron-labeled molecules in the subject to create tomographic images of the location of the radioactivity. In contrast, SPECT supplies tomographic information by rotating detectors around the specimen and collecting only gamma particles that hit the detectors through a collimator (an aperture that allows the detection only of particles that travel directly through the hole). The development of small animal PET and SPECT instrumentation (small animal PET and microSPECT) has made this technology readily accessible for the imaging of biological functions in small living animals (Couturier et al. 2004).
A typical small animal PET scan entails the intravenous (i.v.) or intraperitoneal (i.p.) injection of a positron-emitting imaging probe in the animal. The duration of imaging typically ranges from 5 minutes to 1 hour. A major advantage of PET is its high sensitivity and the ability to quantify the concentration of imaging probes in the body. It is possible to detect radiolabeled molecules at a concentration of 1 pmol per liter. Consequently, the administration of only trace amounts of an imaging probe is necessary for PET imaging and generally does not have pharmacologic effects.
Computed Tomography
Computed tomography (CT) is a technique in which an x-ray source and detector rotate around the specimen in order to produce 3-dimensional serial images and volumetric data with anatomical information about the specimen. The differential absorption of x-rays in the tissue generates contrast. Small animal CT scans can achieve a very high resolution, in the range of a few micrometers. Natural contrast exists between bone and soft tissue, but because contrast between soft tissues is limited, contrast agents (most commonly iodinated compounds) can enhance the ability to visualize tissue vascularization (Ford et al. 2006; Mukundan et al. 2006). However, CT is several orders of magnitude less sensitive than PET and thus requires the administration of contrast agents at much higher doses, which may cause pharmacologic effects in the animals.
Magnetic Resonance Imaging
Magnetic resonance imaging (MRI) uses strong magnetic fields to align the spin of hydrogen nuclei in tissues. The application of a pulse of radio waves "flips" the spin of the hydrogen nuclei into an excited state. The subsequent relaxation of the spin of the hydrogen, to realign with the principal magnetic field, emits a pulse of radio waves that are detected and quantified. MRI reveals information ranging from anatomical structure to chemical composition to physiological parameters such as blood flow (for a review see Beckmann et al. 2001). Successful applications of MRI in the mouse have led to the development of higher-throughput techniques (for a review see McConville et al. 2005). MRI provides a much higher contrast than CT between various soft tissues, and paramagnetic contrast agents can further enhance this contrast (Beckmann et al. 2001).
Optical Imaging
Optical imaging uses one of two methods to generate, and then detect, visible light in the body of a small animal: fluorescence or bioluminescence. The fluorescent method uses fluorophors, which can emit photons after excitation. Fluorescent reporter proteins, such as green fluorescent protein (GFP) and red fluorescent protein (RFP), have been used widely for the labeling of cells or to monitor gene expression. Bioluminescent methods use an enzymatic reaction between the luciferase enzyme and its substrate, a luciferin, to produce light for detection. The most common bioluminescent reporter proteins for imaging in living small animals are the firefly luciferase from Photinus pyralis and the Renilla luciferase from the sea pansy Renilla reniformis.
Sensitive imaging systems enable researchers to detect and quantitate small numbers of cells or organisms that express optical transgenes (Rice et al. 2001), monitor tumor cell growth and regression (Rehemtulla et al. 2000), visualize the kinetics of cell migration (Costa et al. 2001), and look at fluorescent probe activation by enzymes (Ntziachristos et al. 2002). One key technical difference between fluorescence and bioluminescence imaging is that the latter requires the injection of the substrate into the animal before the imaging, whereas no additional animal manipulation is necessary for fluorescence imaging of GFP or RFP expression.
Animal Preparation for Imaging
Depending on the imaging modality and the process under study, animal preparation can range from none (aside from anesthesia; e.g., for CT imaging of bone morphology) to extensive (fasting, anesthesia, and probe injection; e.g., for metabolic imaging with PET tracers). For instance, researchers have noted the effects of animal handling and preparation in small animal PET studies using the glucose analog fluorodeoxyglucose (FDG). Variations in conditions such as fasting and warming alone can greatly influence the distribution of FDG accumulation in the mouse (Fueger et al. 2006); we discuss such impacts in more detail below.
Animal Strain and Gender
One of the initial parameters that may affect results, especially when comparing between experiments, is the use of various strains of mice or rats. Studies show that strain difference alone in mice influences parameters such as the sleep time of anesthesia, stress (Lovell 1986; van Bogaert et al. 2006), immunity, and tumorigenesis (Kolaczkowska et al. 2001; Thaker et al. 2006).
Gender has significant effects on pharmacokinetics, metabolism, and other physiological parameters (Curry 2001; Czerniak 2001). These effects may be due to differences in hormones such as corticosterone, which can influence glucose levels (Champy et al. 2004); in sex hormones (even within a group of female mice), depending on the stage of estrus; and in hepatic enzymes between males and females (Kato and Yamazoe 1992).
Fasting and Diet
Fasting can be an effective way to ensure uniformity in PET results in the clinic as well as in small animal imaging. It is especially useful in FDG imaging to decrease blood glucose levels. In rodents, fasting times of about 6 hours clear the stomach of food; longer fasting, however, may also cause a loss of body mass, a decrease of blood glucose and fatty acid levels, and changes in water intake (Hedrich 2004; Suckow et al. 2006).
Rodent food consumption is tied to the animals' circadian cycle; because they are nocturnal feeders, the removal of food during the dark hours can have a stronger impact on their caloric intake than removal during the light hours. Researchers should therefore take care to control for the length and timing of fasting if it is used in conjunction with imaging studies. It is also appropriate to consider dietary concerns, especially for fluorescence imaging, as food components such as chlorophyll appear to be a significant source of background autofluorescence in mice (McNally et al. 2006).
Circadian Cycles
Rodents are nocturnal animals and so most of their activity takes place during the dark cycle. Research has found that cardiovascular function, blood constituents, gastrointestinal function, and endocrinology also follow a daily cycle (Hedrich 2004). Variation in circadian cycles may mask strain differences in immunological parameters (Kolaczkowska et al. 2001), and rodents' response to pharmacological treatment varies depending on the timing of the treatment during the circadian cycle (Levi 2002). Random interruptions in an animal's circadian cycle can skew the results of imaging studies that compare animal physiology. In imaging experiments that followed the expression of luciferase using optical imaging in mice, researchers found that even viral gene promoter activity is responsive to the circadian cycle (Collaco and Geusz 2003; Levi 2002). It is therefore critical to control the timing of imaging experiments and treatment in order to ensure reproducibility between imaging experiments both in longitudinal studies in a single animal and in studies between groups of animals.
Anesthesia
Anesthesia is often necessary in imaging experiments in order to ensure the constant restraint of the animals. However, with small animals the use of anesthesia may provide some challenges. We review the physiological effects of commonly used anesthetic agents that may confound the results of imaging studies. Besides the specific issues addressed in the next sections, we note that anesthesia in general causes hypothermia in small animals, so it is important to provide appropriate warming during the imaging procedure (Fueger et al. 2006).
Barbiturates
Barbiturates can be classified by their lipid solubility and thus their duration of action; small animal imaging studies most commonly use the short-acting and ultrashort-acting types, which are highly lipid soluble and enter the brain rapidly. As the drugs are cleared by liver metabolism and excreted into the urine, they are redistributed from the central nervous system (CNS) to the general circulation, lowering the animal's level of anesthesia.
Pentobarbital, a short-acting barbiturate, is the most common for rodent anesthesia. The recommended dosage for rats and mice is 40 to 50 mg/kg (Flecknell 1996); depending on the species, strain, and sex, this range of doses results in 15 to 60 minutes of anesthesia and a total sleep time of 120 to 200 minutes (Figure 1; Flecknell 1996; Lovell 1986). The main negative effects of pentobarbital include respiratory depression and cardiovascular effects such as reduced blood pressure, stroke volume, and hypotension (Kohn et al. 1997).
Figure 1 Sleep time of pentobarbital anesthesia in different strains and sexes of mice. This basic study exemplifies how strain and gender choice can influence small animal studies. The significant differences in sleep time may be due to differential expression of metabolic enzymes. Adapted from Lovell DP. 1986. Variation in pentobarbitone sleeping time in mice. 1. Strain and sex differences. Lab Anim 20:85-90.
Ketamine
Ketamine is one of the most commonly used anesthetics in veterinary medicine, most likely because of its large safety margin and compatibility with other drugs (Hau and Van Hoosier 2003). In contrast to other anesthetics, ketamine does not depress respiration or cardiac output. But it has indirect sympathomimetic effects and increases norepinephrine plasma levels and thus may interfere with studies that involve the sympathetic nervous system or experiments that use the norepinephrine transporter as a reporter gene (Anton et al. 2004).
Ketamine is a highly lipid soluble that readily crosses the blood-brain barrier and then redistributes to the circulation as it is metabolized in the liver by cytochrome P450. The common dosage of ketamine is an intraperitoneal (i.p.) injection of 80 to 200 mg/kg for mice and rats (Kohn et al. 1997). At this dose it produces anesthesia for 30 minutes but also induces muscle rigidity, which is commonly treated with the injection of a sedative such as xylazine (a thiazol derivative and alpha 2 adrenergic antagonist). Typically, a 10:1 ratio of ketamine to xylazine produces anesthesia for 20 to 30 minutes and a total sleep time of 60 to 120 minutes in the mouse (Flecknell 1996). It may also induce mild to moderate respiratory depression, hypotension, bradycardia, and hypothermia, as well as hyperglycemia due to xylazine-blocked insulin secretion on pancreatic islets (Abdel el Motal and Sharp 1985).
Inhalation Anesthesia
Inhalation anesthesia involves delivery of the anesthetic via the respiratory system. Although the safe and reliable use of inhalation anesthesia requires extra equipment, its use is increasing in veterinary and research settings because it allows better control over the length and depth of anesthesia than injectable anesthetics.
Probably the most commonly used agent for inhalation anesthesia is isoflurane, usually delivered at 3.5% to 4.5% gas in oxygen to induce anesthesia, which is then maintained with a concentration of 1.5% to 3% isoflurane (Flecknell 1996). Isoflurane has high lipid solubility and is rapidly absorbed through the alveoli into the blood stream and then into the brain. Generally, cardiac function is better maintained with isoflurane than with the injectable anesthetics, although depression of respiration still occurs. There is typically good muscle relaxation with isoflurane and animals recover rapidly from the anesthesia (Flecknell 1996).
Influence of Anesthesia on Small Animal Imaging
Research by Momosaki and colleagues (2004) demonstrated how anesthetic agents can modify the binding of receptor ligands to dopamine D1 receptors in the rat brain. Using a stereotaxic device designed to allow PET imaging of conscious rats, they compared the binding of a carbon-11-labeled dopamine D1 receptor antagonist in the striatum of rats that were either left conscious or anesthetized with ketamine, pentobarbital, or chloral hydrate. Ketamine and chloral hydrate (Scheibler et al. 1999) significantly increased binding of the probe in the striatum as compared to the conscious control animals, while pentobarbital significantly decreased probe binding compared to the control animals. The study did not look at the mechanism of action, but the authors hypothesize that physiological parameters such as blood flow could have affected delivery of the tracer to the tissue or that the anesthetics themselves affected D1 receptor binding affinity.
The glucose analog FDG is a marker of cellular glucose utilization and is widely applied for PET studies in cardiology, neurology, and oncology. Several reports have indicated that some anesthetic agents can have profound effects on glucose metabolism in mice and thus significantly affect the results of FDG-PET studies.
Toyama and colleagues (2004) performed a side-by-side comparison of anesthesia effects on FDG uptake in the mouse brain and heart. They injected FDG intravenously (i.v.) into nonfasting mice that were either conscious or anesthetized with ketamine/xylazine (K/X1) or isoflurane. They found that the isoflurane-anesthetized animals showed significantly lower brain uptake than either the awake animals or the K/X-anesthetized animals. Furthermore, the isoflurane-anesthetized mice had higher heart uptake than the awake animals, whereas the K/X animals had significantly lower heart uptake than the awake animals.
Lee and colleagues (2005) determined the effect of K/X and pentobarbital anesthesia on FDG biodistribution in C57BL6 mice with subcutaneous Lewis lung carcinoma tumors. FDG concentrations in the tumors and in various normal tissues were measured 45 minutes after i.v. injection of FDG and compared with mice that were awake during the uptake period. The experiments took place after the groups of mice fasted for 4 or 20 hours. After 4 hours of fasting, K/X and pentobarbital anesthesia significantly increased the FDG concentration in the blood and decreased the FDG concentration in the myocardium and skeletal muscle. Tumor FDG uptake was not affected, but tumor-to-blood ratios significantly declined for both types of anesthesia. K/X anesthesia significantly decreased plasma insulin levels and increased blood glucose levels to 418 mg/dl. Pentobarbital anesthesia had no effect on blood glucose levels but caused a fivefold increase in insulin levels. After 20 hours of fasting the blood glucose levels decreased to about 75 mg/dl in both control and anesthetized mice; in addition, there was an almost fourfold increase in tumor FDG uptake and a significantly improved contrast between tumor and normal tissues on PET studies. The authors concluded that K/X anesthesia markedly elevates blood FDG activity and reduces tumor uptake ratios through inhibition of insulin release in mice that fasted for 4 hours, whereas pentobarbital induces a similar but less severe response through insulin resistance. These metabolic effects were substantially attenuated after 20 hours of fasting. Hence both the choice of anesthetic and the duration of fasting have important effects on FDG kinetics and PET images of tumor-bearing mice and should be considered when such studies are performed.
Fueger and colleagues (2006) have further investigated the impact of fasting and anesthesia on FDG biodistribution. Their study evaluated the effect of ambient temperature on FDG biodistribution. Groups of severe combined immunodeficient (SCID) mice were first imaged by small animal PET with free access to food, at room temperature (20°C), with no anesthesia during the uptake period (reference condition). The researchers subsequently evaluated the impact on FDG biodistribution of (a) fasting for 8 to 12 hours, (b) warming the animals with a heating pad (30°C), and (c) general anesthesia using isoflurane or ketamine/xylazine. Subcutaneously implanted human A431 epidermal carcinoma and U251 glioblastoma cells served as tumor models. Under the reference condition, there was intense FDG uptake by brown adipose tissue (Figure 2), indicating that this organ was activated to stabilize the body temperature of the mice. Warming significantly reduced the intense FDG uptake by brown adipose tissue and improved visualization of tumor xenografts. Fasting the animals further improved the visualization of tumors. Depending on the study conditions, FDG uptake by normal tissues increased threefold for skeletal muscle; for brown adipose tissue the uptake was 13 times greater, and 15 times more for the myocardium. Tumor FDG uptake increased fourfold and tumor-to-organ ratios were increased up to 17 times by warming and fasting the animals before FDG injection. As in the study by Lee and colleagues (2005), K/X anesthesia caused marked hyperglycemia. Isoflurane anesthesia only mildly increased blood glucose levels and had no significant effect on tumor FDG uptake. Isoflurane markedly reduced FDG uptake by brown adipose tissue and skeletal muscle but increased the activity concentration in the liver, myocardium, and kidney (Figure 2).
Figure 2 Comparison of the changes in FDG uptake in different tissues in the mouse using different animal handling conditions. This study illustrates the importance of understanding the likely physiological effects of animal handling for imaging. Basic conditions such as fasting, warming, and the type of anesthesia greatly influence the biodistribution of PET tracers such as FDG. For all conditions except D, E, and I, the animals were anesthetized for 5 minutes before the injection of FDG; for condition I, the animal was anesthetized with ketamine before injection. Standardized uptake value (SUV) is a measure of the radioactivity and in this image the grey color scale represents the highest concentrations of radioactivity in solid grey (2.5 on the scale, seen in organs such as the bladder) and low radioactivity in light grey (0.0 on the color bar; the "ghostly" image of the mouse). (A) Not fasted, warmed, no anesthesia. (B) Fasted, not warmed, no anesthesia. (C) Fasted, warmed, no anesthesia. (D) Fasted, warmed, no anesthesia, conscious injection. (E) Reference conditions: not fasted, not warmed, no anesthesia. (F) MicroCT, sagittal view for anatomical reference. (G) Not fasted, warmed, isoflurane. (H) Fasted, warmed, isoflurane. (I) Fasted, warmed, ketamine. Reprinted by permission of the Society of Nuclear Medicine from Fueger BJ, Czernin J, Hildebrandt I, Tran C, Halpern BS, Stout D, Phelps ME, Weber WA. 2006. Impact of animal handling on the results of 18F-FDG PET studies in mice. J Nucl Med 47:999-1006 (Figure 1). The original figure is in color and is available both in the online posting of this article and in Fueger et al. (2006).
Recently Dandekar and colleagues (2007) investigated the reproducibility of small animal PET studies using FDG. Notably, in this study the effect of anesthesia on glucose differed from the results reported by Fueger et al. (2006) or by Lee et al. (2005). Using nude mice and isoflurane anesthesia, this group found that glucose levels decreased over the 2-hour period that the animals were monitored. This difference in the animal response could be due to the differences in the sex and strain of the mice. The results might also be a reflection of different animal handling conditions, such as intravenous injection of FDG or different fasting lengths.
These studies with FDG illustrate the importance of understanding the likely physiological effects of anesthesia, standardizing methods, and reporting all animal handling procedures, including the amount and type of anesthesia. To improve tumor visualization, mice should be fasted and warmed before FDG injection and during the uptake period. Isoflurane appears well suited for anesthesia in tumor-bearing mice, whereas K/X should be used with caution as it may induce marked hyperglycemia.
Respiration and Circulation
Even under anesthesia, an animal's respiration and cardiac function can contribute to motion artifacts that may complicate the imaging of the heart and lungs as well as of tumors in the chest.
The monitoring of respiration and circulation in small animals is challenging. The rodent heart rate exceeds 250 beats per minute, and the small lung size makes artificial respiration technically difficult. To monitor these parameters it is necessary to use special electrocardiograms (ECGs) and respiratory monitors specifically designed for small animals. In imaging, electrodes must be placed outside regions of interest to prevent artifacts; and for MRI, the equipment must not be ferromagnetic. Furthermore, because the magnetic field in MRI can produce artifacts in the ECG data, specialized equipment is necessary to synchronize the heart and respiratory data with the imaging data (a method called "gating"). Researchers developing tools for gating have used software and programming to coordinate signals and reduce noise in both MR and PET images of cardiac function and respiration (Cassidy et al. 2004; Lee et al. 2005; Sabbah et al. 2007; Yang et al. 2005). Other approaches include the development of monitoring systems that are impervious to the electromagnetic interference generated in MR studies (Brau et al. 2002). Although small animal intubation is difficult, Namati and colleagues (2006) used it to their advantage when they developed a gating system for studying the lung by CT. They created a technique called intermittent iso-pressure breath hold to "freeze" the animal's breathing during imaging. This gating regimen resulted in images with 2 to 3 times better resolution than those produced using standard techniques.
Because anesthesia affects many aspects of an animal's physiology, researchers have also looked at MR and PET imaging of conscious animals. Most of this work focuses on brain function in the rat due to logistical issues (e.g., animal size, spatial resolution, and sensitivity of imaging devices). MRI stereotaxic devices are now available to restrain the body and head of a conscious rat during retrieval of functional MR images (Khubchandani et al. 2003). There is also a stereotaxic device for use in PET imaging of the rat brain (Momosaki et al. 2004). In both cases, animals had parts of the device implanted in the skull and required conditioning for up to 14 days to tolerate the devices so that they remained calm for imaging. Woody and colleagues (2004a) have reported another approach, called "RatCAP" (rat conscious animal PET), a device that consists of a tiny PET array that fits around the rat's head. There is still an invasive attachment of part of the device to the animal, but the animal can walk around in an enclosure during imaging (Figure 3). This group has also gone on to design a small animal combined PET and MRI based on the RatCAP, which could allow for conscious imaging of the rat brain on both a physiological level (PET) and anatomical level (MRI) in an animal under identical conditions (Woody et al. 2007).
Figure 3 Examples of devices for conscious animal imaging. Because anesthesia can affect many aspects of an animal's physiology, researchers have looked at different devices to image conscious animals using small animal PET and MRI. (A) Rat conscious animal PET (RatCAP). A mini-tomograph with an array of 12 detectors is mounted on the head of the rat, and the rat moves freely in a specialized cage. Reproduced with permission from Woody C, Dzhordzhadze V, Fontaine R, Junnakar S, Kandasamy A, Kriplani A, Krishnamoorthy S, Lecomte R, O'Connor P, Page C, Pratte J-F, Purschke M, Radeka V, Rampil I, Schlyer D, Shokouhi S, Southekal S, Stoll S, Vaska P, Villanueva A, Yu B. 2004. The RatCAP conscious small animal PET tomograph. Nuclear Science Symposium Conference Record, IEEE 4:2334-2338. (B) Rat-sized stereotaxic device made of MRI compatible material. The animal is restrained and can be imaged while conscious. Reproduced with permission from Khubchandani M, Mallick HN, Jagannathan NR, Mohan Kumar V. 2003. Stereotaxic assembly and procedures for simultaneous electrophysiological and MRI study of conscious rat. Magn Reson Med 49:962-967.
Effects of the Imaging Agent and Repeated Imaging on Animal Physiology
Properties such as pH level, tonicity, stability, type of solvent, sterility, viscosity, and rate of absorption are important considerations for any substance injected in animals (Hau and Van Hoosier 2003; Morton et al. 2001; Woody et al. 2004b). The general recommendation for the maximum dose of intravenous injections is 4% to 5% of the animal's blood volume (i.e., an approximate total injection volume of <200 μl for a mouse and 1,000 μl for a rat), and the maximum intraperitoneal injection volume is 10 mL/kg body mass (Hau and Van Hoosier 2003; Hedrich 2004; Kohn et al. 1997; Morton et al. 2001). For imaging probes labeled with short-lived radioisotopes, the injection volume increases quickly over time. For example, consider an imaging probe labeled with the positron-emitting radionuclide carbon-11, which decays with a half-life of 20 minutes. When the initial radioactivity concentration after production is 10 mCi/ml, an injection of only 20 μl is necessary to administer a typical dose of 200 μCi to the first mouse being studied. However, 2 hours later an injection of 1,280 μl would be necessary to achieve the same amount of radioactivity. Therefore, there exists a maximum time after a radiolabeled tracer has been produced in which it can be used in animals.
A related problem is the "mass effect." Initially, radiolabeled agents are prepared with a high specific activity (radioactivity per unit mass of substance). Therefore, only minuscule trace amounts of the labeled ligand are injected and a low binding occupancy of receptors is achieved. This prevents the occurrence of a pharmacological effect of the tracer. However, with radioactive decay the specific activity decreases and the number of molecules injected increases. This problem is aggravated by the fact that, relative to body weight, PET imaging agents are generally injected at much higher amounts in mice than in humans. This higher amount of radioactivity is necessary to achieve a sufficient spatial resolution for the imaging of mice. For example, the typical dose for a human PET study is approximately 10 mCi of fluorine-18. If the dose were scaled down by body weight, a 25-gram mouse would be injected with only 0.0036 mCi (10 mCi * 25 g / 70,000 g). However, the actual dose is about 0.2 mCi, more than 50 times higher. For many probes, such as FDG or other metabolic substrates, this does not present a problem because the amount injected is still far below the threshold of pharmacologic effects. For some high-affinity receptor ligands, however, pharmacologic effects may occur (Kung and Kung 2005). Because small animal SPECT is less sensitive than small animal PET and because typical SPECT imaging probes show a lower specific activity than PET imaging probes, mass effects are of greater concern for SPECT studies.
Volume may also be an issue for CT contrast agents (Ford et al. 2006; Mukundan et al. 2006). Most iodinated CT contrast agents clear the rodent circulation quickly, hence a specialized formulation or constant infusion is necessary. Furthermore, the concentration of iodine in the formulation determines the resulting level of contrast resolution; thus formulations with less iodine require higher volumes to reach the same level of contrast as those with more iodine. For these reasons complications such as renal toxicity (Gale et al. 1984) and hypersensitivity reactions (Gueant-Rodriguez et al. 2006) are a risk when using iodinated compounds in rodents.
Although studies have not reported adverse reactions to the mass quantities of luciferase substrates injected in mice, complications from repeated injection or from poor preparation of the substrates could affect small animal studies. For optical bioluminescence imaging, it is usually possible to adjust the injection volume according to the route of administration (i.v. or i.p.) and thus reduce the risk of volume overload. Interestingly, researchers have found it feasible to image mice by putting luciferase substrates in their drinking water (Hiler et al. 2006), a less stressful way to deliver the substrate.
Radiation Exposure
PET and SPECT
The fact that small animal PET requires the injection of radionuclides raises the question of how much radiation dose the animal receives. As discussed above, the tissue activity concentration in rodent studies is markedly higher than in humans and results in a higher radiation dose from electron or positron radiation. On the other hand, the mouse's small size allows a larger fraction of gamma radiation to escape. For these reasons, an estimate of the radiation dose is not straightforward, and radiation dosimetry studies on small animals are still rare.
Funk and colleagues (2004) calculated the radiation dose of a rodent using Monte Carlo simulation of the rodent as a homogeneous ellipsoid. They estimated a whole body dose of 6 to 90 centigrays (cGy) for mice and 1 to 27 cGy for rats based on radioisotopes typically used in PET or SPECT, such as 18F, 111In, or 125I. In comparison to the reported lethal dose of ionizing radiation in mice of 6.5 to 7 Gy, the simulated exposure may seem low. However, this type of whole body dose calculation does not take into account the biodistribution of the radionuclide, but rather assumes that the absorbed energy is evenly distributed over the entire body. Thus this approach typically underestimates the radiation risk to areas of high radionuclide concentration and provides only a rough estimate of radiation exposure.
Taschereau and Chatziioannou (2007) used Monte Carlo simulations of an anatomically defined mouse phantom to calculate more realistic radiation dose estimates for PET imaging probes. The absorbed dose for various tissues after injection of 200 uCi FDG ranged from 19 milligrays (mGy) for skin to 4,000 mGy for the bladder wall. The large range in radiation dose is due to the fact that some tissues (e.g., skin) accumulate little radioactivity, some (e.g., tumors, the brain and heart) accumulate tracer, and others (e.g., the kidney and bladder) are involved with the clearing of the tracer from circulation and receive higher radiation doses. Although the doses calculated are lower than the lethal doses reported, they are more than 10 times higher than for human PET studies. Biological effects have been reported to occur with ionizing radiation doses of less than 1 mGy (Hooker et al. 2004; Taschereau et al. 2007), and FDG given at 10 times the normal mouse imaging dose has been used in a mouse model of breast cancer to attenuate tumor growth (Moadel et al. 2005).
CT
Although the radiation levels in microCT are generally not lethal to the animal, higher-resolution studies can result in greater amounts of energy absorbed by the animal, leading to "invasive" levels. According to a study by Taschereau and colleagues (2006), small animal CT causes radiation doses ranging from 70 mGy to 400 mGy. In general, bony structures received a higher dose than soft tissues. The radiation dose experienced by the animal is a factor of the desired resolution, the energy of the x-ray used, the duration of the scan, and the number of serial scans of the animal. The use of different scan protocols (e.g., an ultrafast protocol) can significantly reduce the radiation exposure of mouse tumors to x-rays (Carlson et al. 2007; Taschereau et al. 2006). But concurrent use of PET or SPECT can also increase the animal's total dose. Taschereau and colleagues estimated that a longitudinal study consisting of five FDG-PET and CT scans in a single animal could equal a total exposure of 1 gray (Gy) for that animal. Recalling that 6.5 to 7 Gy is a lethal dose for mice, and that even lower doses have shown biological effects, investigators should be aware of the potential interference of imaging, especially when a high radiation dose is given to the organ or structure under investigation and when longitudinal studies are performed.
Repeated Bioluminescence Imaging
Recent work by Brutkiewicz and colleagues (2007) has indicated that the expression of the bioluminescent reporter firefly luciferase has an effect on tumor growth. Although the researchers did not fully investigate the exact mechanism by which high luciferase-expressing tumors experience growth inhibition, they did find that relatively high luciferase-expressing xenografts, when repeatedly imaged, showed slower growth than xenografts with low luciferase expression levels. This type of effect of luciferase imaging in tumor cells has not been reported previously and, as with the effects of radiation dose, it is another concern that warrants attention when performing serial imaging in animals expressing luciferase.
Stress
Unintentional physical or mental stress on animals can affect experimental results. Longitudinal imaging studies create numerous occasions for animals to be stressed. Some of the factors to consider include repeated injection, handling, transportation, experimental conditions (tumor burden or surgery), hypothermia, and fasting. Acute stress conditions, such as those that result from restraint and injection, release epinephrine and corticosterone, immediately increasing glucose levels and heart rate and inducing hyperthermia (Brutkiewicz et al. 2007; Meijer et al. 2005, 2006; Suckow et al. 2006).
Conclusions
There are many animal handling issues to consider in small animal imaging. It is important to address basic considerations such as the strain and sex of the animal, the pain and distress caused by the experiment, and the time of day that imaging and experimental procedures occur. As demonstrated most clearly with FDG PET, animal preparation and anesthesia can significantly influence the results of imaging studies. Conversely, imaging procedures themselves can affect the results of experiments, as seen in the optical imaging of luciferase-expressing xenografts. In longitudinal small animal imaging studies, control groups may be necessary to exclude such effects, as knowledge of the physiologic and pharmacologic effects of repeated imaging is still limited. To ensure reproducible experimental conditions, the study design should clearly report the strain of the animal, gender, fasting times and lengths, type and dose of anesthesia, and how often individual animals were imaged, and these factors should be consistent throughout the experiment. Further research is necessary to determine how animal handling can be refined to maximize the information obtained from imaging, to minimize the impact of imaging on the studied biological processes, and to improve animal welfare.
Abbreviations used in this article: CT, computed tomography; FDG, fluorodeoxyglucose; K/X, ketamine/xylazine; MRI, magnetic resonance imaging; PET, positron emission tomography; SPECT, single photon emission computed tomography
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