Abstract
Small-animal PET has opened the way for imaging 18F-FDG uptake in murine tumor models, but the need for anesthesia raises concern over its potential influence on 18F-FDG kinetics. We thus investigated such effects on cultured cells and on tumor-bearing mice after short- and long-term fasting. Methods: Lewis lung carcinoma (LLC) cells and cardiomyoblasts were treated for 2 h with a 100 μmol/L concentration of xylazine, ketamine, xylazine plus ketamine (Xy/Ke), or pentobarbital and were measured for 18F-FDG uptake. LLC tumor–bearing C57BL6 mice that had been kept fasting for either 4 or 20 h were injected with Xy/Ke, pentobarbital, or saline and were administered 1.8 MBq of 18F-FDG 15 min later. Biodistribution studies and plasma glucose and insulin assays were performed 45 min after injection. Separate anesthetized and control mice underwent 18F-FDG PET. Results: 18F-FDG uptake in LLC cells was unaffected by anesthetic agents, whereas xylazine and ketamine caused a small increase of uptake in cardiomyoblasts. In mice kept fasting 4 h, Xy/Ke induced a marked elevation of 18F-FDG activity (percentage injected dose [%ID]) in blood (6.8 ± 0.9 %ID/g vs. 1.1 ± 0.6 %ID/g) and kidneys while decreasing myocardial uptake (2.3 ± 1.3 %ID/g vs. 4.7 ± 1.8 %ID/g). Target-to-blood ratios were significantly reduced. Pentobarbital caused a moderate increase in blood activity (2.5 ± 0.8 %ID/g), decreased myocardial uptake (2.8 ± 0.5 %ID/g), and reduced target-to-blood ratios. PET images of mice kept fasting 4 h were consistent with the biodistribution data. Insulin levels were lower with Xy/Ke and higher with pentobarbital. In mice kept fasting 20 h, Xy/Ke and pentobarbital increased blood 18F-FDG activity (5.5 ± 2.2 and 4.9 ± 0.9 %ID/g vs. 2.4 ± 0.3 %ID/g) and reduced target-to-blood ratios, but these changes were substantially attenuated, compared with those in mice kept fasting 4 h. In addition, insulin levels were low and unaffected by anesthesia. Conclusion: Xy/Ke anesthesia markedly elevates blood 18F-FDG activity and reduces tumor uptake ratios through inhibition of insulin release in mice kept fasting 4 h, whereas pentobarbital induces a similar but less severe response through insulin resistance. These metabolic effects, however, are substantially attenuated after 20 h of fasting. Hence both the choice of anesthetic and the duration of fasting have important effects on 18F-FDG kinetics and PET images of tumor-bearing mice and should be considered when such studies are performed.
An augmented demand for glucose use is a fundamental metabolic feature of tumor cells that is exploited by oncologic 18F-FDG PET studies. The use of 18F-FDG PET in cancer patients has now gained widespread acceptance for initial staging of disease, management of recurrent disease, and monitoring of therapeutic response (1). More recently, molecular imaging in small animals has emerged as a powerful technique for investigating disease processes at the molecular and cellular levels. As such, small-animal PET with 18F-FDG provides a unique opportunity to advance our understanding of glucose metabolism in tumors of living mouse models (2–4). Imaging small animals requires anesthesia to prevent motion artifacts. This presents a potential problem for 18F-FDG PET, because various anesthetic agents have been found to affect glucose turnover rates and alter its use in such tissues as the brain (5–10). These findings imply that anesthetic agents may also influence 18F-FDG distribution and PET image results in animal tumor models, but this issue has not yet been elucidated. Moreover, important questions such as relative magnitudes of effect by different anesthetic agents, their potential mechanisms of effect, and the role of fasting duration have not been addressed. In this study, we thus investigated the effects of the widely used anesthetic agents xylazine plus ketamine (Xy/Ke) and pentobarbital on 18F-FDG uptake in cultured cells and on 18F-FDG biodistribution in tumor-bearing mice after a short- or long-term fast.
MATERIALS AND METHODS
Cell Culture and 18F-FDG Uptake Experiments
Lewis lung carcinoma (LLC) cells (established from lung cancer of a C57BL6 mouse) and H9C2 embryonic rat cardiomyoblasts were purchased from the American Type Culture Collection and maintained in Dulbecco’s modified Eagle medium (Gibco BRL) supplemented with 10% fetal bovine serum, 2 mmol of glutamine per liter, 100 U of penicillin per milliliter, and 100 mg of streptomycin per liter at 37°C in 5% CO2. Cardiomyoblasts were differentiated by being cultured in medium with 2% fetal bovine serum for 3 d, followed by incubation in 0.5% fetal bovine serum starting the day before the experiments. The experiments were performed on 70%–80% confluent cells in 12-well plates. The cells were treated with a 100 μmol/L concentration of xylazine, ketamine, Xy/Ke, or pentobarbital added to the culture medium for 2 h, followed by incubation with 1.8 MBq of 18F-FDG at 37°C and 5% CO2 for 45 min, then rapidly washed twice with phosphate-buffered saline and measured for radioactivity on a high-energy γ-counter (Wallac).
Animal Tumor Model Preparation
All animal procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (11) and were approved by the Institutional Committee on Animal Care and Use. Tumors were grown in male C57BL6 mice by subcutaneous injection of 2 × 107 LLC tumor cells into the right flank. Biodistribution experiments were performed when tumor diameter reached approximately 2 cm.
Anesthesia
All experiments began in the late morning; food had been withheld from the animals since early morning (4 h of fasting) or the evening before (20 h of fasting). Water was allowed ad libitum. On the day of the experiment, tumor-bearing mice were given an intraperitoneal injection of Xy/Ke (9 and 110 mg/kg) or pentobarbital (50 mg/kg). Fifteen minutes later, the animals were checked for depth of anesthesia by a toe pinch to verify no response and then were injected with 18F-FDG. Control animals were intraperitoneally injected with saline, injected with 18F-FDG by immobilization in a restrainer, and placed in a quiet and dark cage until they were sacrificed.
18F-FDG Biodistribution and Glucose and Insulin Measurements
A 1.8-MBq injection of 18F-FDG mixed with isotonic saline to a total volume of 150 μL was administered through a tail vein. After the 45-min 18F-FDG uptake period, the animals were sacrificed by cervical dislocation, and major organs were promptly extracted, washed, weighed, and measured for radioactivity on a γ-counter. Organ and tumor uptake levels were expressed as mean ± SD percentage injected dose (%ID) per gram of tissue. The plasma of each animal was assayed for glucose concentration using a dedicated glucose analyzer (2300 STAT Plus; YSI Life Sciences) and for insulin concentration using an immunoradiometric assay kit (INS-IRMA kit; BioSource).
18F-FDG PET Imaging of Tumor-Bearing Mice
LLC tumor–bearing mice were intraperitoneally injected with Xy/Ke, pentobarbital, or saline as above. Fifteen minutes later, a 9.2-MBq injection of 18F-FDG was administered through a tail vein with the mouse under anesthesia or, in the case of saline-injected controls, immobilized in a restrainer. Image acquisition started 45 min after injection and was performed on an Advance PET scanner (GE Healthcare) without attenuation correction. The control animals were sacrificed by cervical dislocation immediately before imaging to prevent motion.
Statistical Analysis
The significance of differences in 18F-FDG uptake levels, organ-to-blood uptake ratios, and blood insulin levels between multiple groups was analyzed by 1-way ANOVA, with P values < 0.05 considered significant. The Bonferroni multiple-comparison test was performed as a post hoc test to determine the significance of the difference between any 2 groups.
RESULTS
18F-FDG Uptake in Cultured Cells Treated with Anesthetic Agents
In cultured LLC tumor cells, none of the anesthetic agents tested affected 18F-FDG uptake levels. In the cardiomyoblasts, minor increases of 18F-FDG uptake were induced by xylazine, ketamine, and Xy/Ke (to 123% ± 1%, 117% ± 4%, and 128% ± 4%, respectively, of controls) but not by pentobarbital (Fig. 1).
Effect of anesthetic agents on 18F-FDG uptake in cultured LLC cells and H9C2 cardiomyoblasts. Uptake was measured after cells had been incubated for 45 min with 18F-FDG and after a 2-h pretreatment with a 100 μmol/L concentration of xylazine, ketamine, Xy/Ke, or pentobarbital (Pentob). Data are mean ± SD of percentage uptake relative to control cells, obtained from triplicate samples of a single experiment representative of 2 separate experiments. *P < 0.01. **P < 0.001.
Effect of Xy/Ke on 18F-FDG Biodistribution in Mice Kept Fasting 4 h
The biodistribution of 18F-FDG in LLC tumor–bearing C57BL6 mice is shown in Table 1. In mice kept fasting 4 h, the effect of Xy/Ke anesthesia was distinguished by a marked, 5.9-fold, increase of blood 18F-FDG activity over control levels. Another prominent finding was a 7.9-fold increase of activity in the kidneys. Liver uptake was also increased 3.8-fold, whereas myocardial uptake was reduced to less than half of control levels. Skeletal muscle uptake was higher in control animals, probably because they were able to move about after 18F-FDG injection. Although Xy/Ke did not significantly affect absolute tumor uptake levels, high blood activity led to a substantial reduction in tumor-to-blood ratios (0.8 ± 0.3 vs. 6.7 ± 3.6) and myocardium-to-blood ratios (0.3 ± 0.2 vs. 4.3 ± 1.3; Fig. 2A).
Effect of anesthetic agents on organ-to-blood 18F-FDG ratio, insulin level, and blood 18F-FDG activity. (A and B) Tumor- and myocardium-to-blood ratios of 18F-FDG uptake in mice that had been kept fasting for 4 h (A) or 20 h (B). (C and D) Plasma insulin levels (μIU/mL) (C) and relationship to blood 18F-FDG activity (D) in mice that had been kept fasting for 4 h (dotted lines were arbitrarily drawn to separate the 3 groups). All bar graphs are represented as mean ± SE. Pentob = pentobarbital. *P < 0.05. **P < 0.01. ***P < 0.001 compared with controls.
18F-FDG Biodistribution, Plasma Glucose, and Insulin Levels Obtained With or Without Anesthesia and After 4 or 20 Hours of Fasting in LLC-Bearing Mice
Effect of Pentobarbital on 18F-FDG Biodistribution in Mice Kept Fasting 4 h
Pentobarbital anesthesia also induced a significant, 2.2-fold, elevation of blood 18F-FDG activity over control levels, although the magnitude was not as pronounced as with Xy/Ke (Table 1). Myocardial uptake was significantly decreased. High blood activity in this group also led to a significant reduction of tumor-to-blood ratios (2.8 ± 0.6) and myocardium-to-blood ratios (1.2 ± 0.4; Fig. 2A).
18F-FDG PET Imaging Results in Mice Kept Fasting 4 h
PET studies of mice kept fasting 4 h revealed images consistent with the biodistribution results. In the nonanesthetized mouse, the subcutaneous tumor and the myocardium were clearly visualized, with high target-to-background contrast. The Xy/Ke- and pentobarbital-anesthetized mice demonstrated significantly inferior tumor contrast, and visualization of the myocardium was poor (Fig. 3).
Coronal whole-body 18F-FDG PET images of mice bearing subcutaneous Lewis lung tumors (arrowheads) in right flank and kept fasting 4 h. Images were obtained on clinical whole-body PET scanner rather than dedicated small-animal PET scanner. Arrow = heart; B = brain; Bl = urinary bladder; control = nonanesthetized mouse; L = liver; pentob = pentobarbital-anesthetized mouse; Xy/Ke = Xy/Ke-anesthetized mouse.
Effect of Anesthesia on Insulin Levels in Mice Kept Fasting 4 h
When we evaluated plasma insulin levels at the time of the biodistribution studies, mice kept fasting 4 h and anesthetized with Xy/Ke had substantially lower insulin levels than did pentobarbital-anesthetized mice (Table 1; Fig. 2C). On a scattergram display, the control group had low blood 18F-FDG activity with variable plasma insulin levels, the Xy/Ke group had markedly increased blood 18F-FDG activity with very low plasma insulin levels, and the pentobarbital group had moderately increased blood 18F-FDG activity with plasma insulin levels that were high in 4 mice and low in 2 mice (Fig. 2D). Those 2 mice showed normal-range plasma glucose levels despite high blood 18F-FDG activity, suggesting that plasma glucose and insulin levels had returned to normal before blood 18F-FDG activity had.
Effect of Anesthesia on 18F-FDG Distribution and Insulin Levels in Mice Kept Fasting 20 h
In mice kept fasting 20 h, Xy/Ke elevated blood 18F-FDG activity 2.2-fold over control levels and increased uptake in the liver and kidneys (Table 1). Interestingly, a mild increase in tumor 18F-FDG uptake was seen in these mice. Pentobarbital injection elevated blood 18F-FDG activity 2.0-fold over control levels but had no effect on tumor uptake (Table 1). Tumor- and myocardium-to-blood ratios were significantly reduced with both anesthetic agents, but less so than in mice kept fasting 4 h (Fig. 2B). Plasma insulin levels during distribution studies were similarly low in all mice kept fasting 20 h, regardless of whether they were administered Xy/Ke, pentobarbital, or saline (Table 1).
DISCUSSION
Xy/Ke and pentobarbital are the most widely used injectable agents to anesthetize rodents for imaging studies. The results of this investigation demonstrated that, in mice kept fasting 4 h, both Xy/Ke and pentobarbital anesthesia induced a substantial increase of blood 18F-FDG activity. The manifest effects of anesthesia on blood glucose or 18F-FDG levels are the result of a complex interaction of multiple mechanisms that include changes in endocrine secretion, hepatic and renal handling, and uptake by peripheral tissues. In our study, altered 18F-FDG biodistribution by Xy/Ke and pentobarbital was accompanied by opposite directions of change in plasma insulin concentrations, indicating different mechanisms of effect.
Xylazine (2-(2,6-xylidino)-5,6-dihydro-4H-1,3-thiazine hydrochloride) is a clonidine analog with sedative, anesthetic, analgesic, and muscle relaxation effects. When ketamine is used, xylazine is routinely added to provide stable sedation and avoid muscle rigidity (12). Our results showed that Xy/Ke markedly increased blood 18F-FDG activity and substantially reduced plasma insulin levels. These findings are consistent with the well-known property of xylazine of stimulating pancreatic α2-adrenergic receptors to block insulin secretion and induce a strong hyperglycemic effect (13,14). Such an effect has been reported in various animal species including dogs (15), rats (16), and recently C57BL6 mice (17). Ketamine is a phencyclidine derivative used for its anesthetic effect of dissociating thalamic and limbic activity from neocortical function. Although increased blood glucose has been associated with the use of ketamine (18), whether it commonly induces hyperglycemia is less well established. Thus, the high blood 18F-FDG activity and glucose concentration for Xy/Ke-injected animals in our study was most likely caused by the hypoinsulinemic effect of xylazine. Because elevated plasma glucose levels can decrease 18F-FDG uptake by competition for transport and metabolism, it is likely that such a decrease contributed to the altered 18F-FDG biodistribution that was observed.
Anesthesia with pentobarbital in mice kept fasting 4 h also led to a significant elevation of blood 18F-FDG activity. However, unlike the insulin-lowering effect of Xy/Ke, pentobarbital caused a prominent increase in plasma insulin levels. The effect of pentobarbital anesthesia on glucose use and glucose tolerance has previously been evaluated in rats by several investigators (19–23). Pénicaud et al. observed in pentobarbital-injected rats a transient elevation of blood glucose at 3 min that returned to basal levels by 40 min (20). Clark et al. (21) and Vera et al. (22) also found mild elevations of plasma glucose in rats injected with pentobarbital, whereas Johansen et al. did not (23). It may be that the observed levels of blood glucose after pentobarbital injection vary with the time of sampling because its induction of hyperglycemia is relatively mild and transient. In our study, increases in blood 18F-FDG activity were not as marked with pentobarbital as with Xy/Ke. Moreover, glucose levels in the pentobarbital group were not elevated despite the significantly higher 18F-FDG activity in the same blood samples. A possible reason may be that 18F-FDG kinetics are not identical to glucose kinetics. Also contributing may be the fact that 18F-FDG was administered 15 min after pentobarbital and had that much less time to return to normal levels than did glucose. The effect of pentobarbital on plasma insulin levels has also previously been studied in rats. Whereas Pénicaud et al. did not observe a significant effect (20), Vera et al. (22) and Johansen et al. (23) found a significant elevation of plasma insulin levels after pentobarbital anesthesia. The hyperinsulinemia induced by pentobarbital implies that its hyperglycemic effect is associated with an insulin-resistant state. Speculative mechanisms for this phenomenon include stimulated secretion of growth hormone, which can induce insulin resistance (24), and a direct inhibitory effect on glucose transporters (25,26).
In our study, the reduced tumor 18F-FDG uptake ratios and PET image contrast caused by both Xy/Ke and pentobarbital anesthesia were due to high blood activity rather than to differences in absolute tumor uptake levels. The results of our in vitro uptake experiment also indicated no direct effect on 18F-FDG uptake by tumor cells, although an effect of anesthetic agents on 18F-FDG metabolism might have caused a difference in the relative contribution of unmetabolized 18F-FDG to net 18F-FDG uptake. On the other hand, Xy/Ke and pentobarbital both significantly decreased absolute myocardial 18F-FDG uptake and caused poor visualization of the heart on PET images. A similar effect has previously been observed on 18F-FDG small-animal PET images of Xy/Ke-anesthetized normal BALB/c mice, although the authors of that study did not obtain biodistribution data (9). No evidence suggests that xylazine or pentobarbital may directly interfere with glucose uptake by cardiomyocytes. This lack of interference is also supported by the results of our cultured cardiomyoblast experiments, in which no reduction of 18F-FDG uptake by the anesthetic agents was observed. However, both Xy/Ke anesthesia (27–30) and pentobarbital anesthesia (29,30) are known to markedly depress heart rate and left ventricular ejection fraction in mice. Hence, the reduction of 18F-FDG uptake in the myocardium of mice anesthetized by Xy/Ke or pentobarbital is most likely an indirect effect of depressed cardiac contractility.
For the Xy/Ke group, another prominent feature was a marked increase of 18F-FDG activity in the kidneys, likely related to the high urinary bladder activity seen on the PET images. In rats, xylazine anesthesia is known to enhance urinary flow rate and urinary sodium excretion (31). This diuretic and natriuretic response is thought to result from the inhibitory effect of xylazine on vasopressin secretion and the direct vasopressin antagonistic action of xylazine on the distal nephron (32,33). Hence, the high renal 18F-FDG activity caused by Xy/Ke anesthesia appears to be due to a combination of blood activity–elevating effect and diuretic effect. This high renal activity may be a disadvantage of Xy/Ke anesthesia for 18F-FDG PET of small animals when the region of interest is near the kidneys or bladder.
A potential limitation of our PET experiment is that the control mouse had to be sacrificed immediately before PET acquisition to prevent motion whereas the anesthetized animals were imaged while alive. However, because the PET acquisition lasted only 5 min, the image result is not likely to have significantly been affected.
For 18F-FDG PET studies of mice, food is generally withheld starting the morning of the experiment, usually resulting in fasting durations of approximately 3–4 h. However, to further reveal the impact of fasting duration on the effects of anesthetic agents on 18F-FDG biodistribution, we extended our investigation to mice kept fasting 20 h. In these mice, we found that the elevating effects of both Xy/Ke and pentobarbital on blood 18F-FDG activity were substantially attenuated, compared to the effects in mice kept fasting 4 h. Furthermore, the anesthetic agents did not affect the plasma insulin levels of mice kept fasting 20 h.
CONCLUSION
In conclusion, the present study demonstrated that in tumor-bearing mice kept fasting short-term, both Xy/Ke anesthesia and pentobarbital anesthesia resulted in a significant elevation of blood 18F-FDG activity. The Xy/Ke-induced effects were greater, probably through the mediation of inhibited insulin secretion, and were accompanied by markedly increased renal uptake. Pentobarbital-induced effects were less severe than Xy/Ke-induced effects and appeared to be mediated through an insulin resistance–like state. The metabolic effects of these anesthetic agents, however, were minimized when mice had been kept fasting longer. Hence, both the choice of anesthetic and the duration of fasting are important factors that can affect biodistribution and image results in 18F-FDG PET of tumor-bearing mice and should be considered in such studies.
Acknowledgments
We thank Hyuk Lee for technical assistance with PET. This work was supported by the Korea Research Foundation Grant KRF-2005-202-E00116. This study was presented in part at the 52nd annual meeting of the Society of Nuclear Medicine, Toronto, Ontario, Canada, June 18–22, 2005.
Footnotes
Received Apr. 21, 2005; revision accepted May 26, 2005.
For correspondence contact: Kyung-Han Lee, MD, Department of Nuclear Medicine, Samsung Medical Center, 50 Ilwondong, Kangnamgu, Seoul, Korea. E-mail: khleenm{at}yahoo.co.kr
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