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Original Article
Normal Values of Thyroid Uptake of 99mTechnetium Pertechnetate SPECT in Mice with Respect to Age, Sex, and Circadian Rhythm Nicola Beindorff1; Annika Bartelheimer2,3; Kai Huang3; Mathias Lukas3; Catharina Lange3; Eleonore L. Huang3; Jörg R. Aschenbach2; Janet F. Eary4; Ingo G. Steffen3*; Winfried Brenner3* Experimental Radionuclide Imaging Center (BERIC), Charité – Universitätsmedizin Berlin, Germany;
2Institute
of Veterinary Physiology, Department of Veterinary Medicine, Freie Universität Berlin, Germany;
3Department 4US
of Nuclear Medicine, Charité – Universitätsmedizin Berlin, Germany;
National Institutes of Health/NCI/DCTD
Keywords Age, sex, circadian rhythm, SPECT, technetium uptake, thyroid, estrous cycle
Summary Aim: The aim of this study was to establish normal values for thyroid uptake of 99mtechnetium pertechnetate (99mTcO4) as a function of age, sex and circadian rhythm in mice. Methods: In 12 female (F) and 12 male (M) C57BL/6N mice, nine consecutive SPECT images of 10 min duration each were acquired as dynamic acquisitions beginning 5 min after intravenous injection of 80 MBq 99mTcO . Each mouse was imaged in follow4 up studies up to 24 months (A: 1 month; B: 3 months; C: 6 months; D: 12 months; E: 24 months). In order to assess for physiologic changes related to circadian rhythm, animals were imaged during light (sleeping phase, SP) as well as during night conditions (awake phase, AP). The percentage tracer uptake of the injected activity is expressed as median %ID. Correspondence to Professor Dr. Winfried Brenner Department of Nuclear Medicine Charité – Universitätsmedizin Berlin Augustenburger Platz 1 13353 Berlin Germany E-mail:
[email protected] Tel: +49–30/450527051 Fax: +49–30/4507 27051
* Both senior authors contributed equally to this work.
© Georg Thieme Verlag KG 2018
Results: Female mice showed significantly higher uptake than males (F 1.6, M 1.1; p < 0.001). This effect was observed up to the age of 12 months: A (F 1.6, M 1.1; p < 0.001), B (F 1.7, M 1.1; p < 0.001), C (F 1.8, M 1.2; p < 0.001), D (F 1.6, M 1.2; p < 0.001), E (F 1.1, M 1.1; p = 0.79). Impact of age on uptake could be observed in females (p = 0.056) and was not present in males (p = 0.27). A significant effect of circadian rhythm could not be observed in females (SP 1.6, AP 1.7; p = 0.65) but in males (SP 1.2, AP 1.1; p = 0.02). Conclusion: This study showed a significant influence of sex on thyroid 99mTcO4 uptake in mice. Sex was also a significant factor affecting age-related changes in uptake in female mice but not in males. In contrast, circadian rhythm had no relevant impact on 99mTcO4 uptake. Therefore, design of thyroid uptake studies in mice using 99mTcO4 should consider animal sex, and in females, age as significant factors affecting uptake.
Normwerte für den Schilddrüsen Uptake von 99mTechnetium-Pertechnetat in Abhängigkeit von Alter, Geschlecht und circadianem Rhythmus bei der Maus Nuklearmedizin 2018; 57: 181–189 https://doi.org/10.3413/Nukmed-0978-18-05 received: May 28, 2018 accepted: August 8, 2018
Schlüsselwörter Alter, Geschlecht, circadianer Rhythmus, SPECT, Technetium Uptake, Schilddrüse, ovarieller Zyklus
Zusammenfassung Ziel: Ziel dieser Studie war die Erhebung von Normwerten für den Schilddrüsen Uptake von 99mTechnetium-Pertechnetat (99mTcO ) in Ab4 hängigkeit von Alter, Geschlecht und circadianem Rhythmus bei Mäusen. Methoden: Bei 12 weiblichen (F) und 12 männlichen (M) C57BL/6N Mäusen wurden neun konsekutive SPECT Akquisitionen von jeweils 10 min Dauer 5 min nach intravenöser Injektion von 80 MBq 99mTcO4 durchgeführt. Jede Maus wurde mehrmals bis zu 24 Monaten untersucht (A: 1 Mo; B: 3 Mo; C: 6 Mo; D: 12 Mo; E: 24 Mo). Um Veränderungen auf Grund des circadianen Rhythmus zu erfassen wurden die Tiere während der Hellphase (Schlafphase, SP) sowie während der Dunkelphase (Wachphase, AP) untersucht. Der prozentuale Tracer Uptake ist als Median %ID der injizierten Aktivität aufgeführt. Ergebnisse: Weibchen zeigten einen signifikant höheren Uptake als Männchen (F 1,6, M 1,1; p < 0,001). Dieser Effekt wurde bis zu einem Alter von 12 Mo beobachtet: A (F 1,16, M 1,1; p < 0,001), B (F 1,7, M 1,1; p < 0,001), C (F 1,8, M 1,2; p < 0,001), D (F 1,6, M 1,2; p < 0,001), E (F 1,1, M 1,1; p = 0,79). Ein altersabhängiger Einfluss zeigte sich bei Weibchen (p = 0,056), jedoch nicht bei Männchen (p = 0,27). Ein signifikanter Effekt des circadianen Rhythmus konnte bei Männchen (SP
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1Berlin
Beindorff N et al.: Thyroid uptake of 99mTc-pertechnetate SPECT in mice
1,2, AP 1,1; p = 0,02) nicht aber bei Weibchen (SP 1,6, AP 1,7; p = 0,65) beobachtet werden. Schlussfolgerung: Diese Studie zeigt einen signifikant geschlechtsabhängigen Einfluss auf den Schilddrüsen Uptake von 99mTcO4 bei Mäusen. Im Gegensatz zu Männchen treten bei Weibchen altersabhängige Veränderungen im Uptake auf. Dagegen hat der circadiane Rhythmus keinen relevanten Einfluss. Folglich sollte bei dem Design von Schilddrüsenuntersuchungen mit 99mTcO4 bei der Maus Geschlecht und bei weiblichen Tieren auch Alter als signifikante Faktoren berücksichtigt werden.
Introduction In addition to standard laboratory tests, thyroid scintigraphy is one of the mainstays for evaluating thyroid function. Quantification of thyroid radiopharmaceutical uptake allows local and global function assessment reported as the accumulation and the biokinetics of radioiodine and 99mtechnetium pertechnetate (99mTcO4) within the gland (5). 99mTcO4 serves as a substitute for radioiodine and is a widely used test in clinical and preclinical thyroid gland imaging due to low cost and wide availability (9, 38). Thyroid 99mTcO4 uptake is mediated by the sodium-iodide symporter (NIS) that actively transports several anions into thyroid follicular cells and other extra-thyroidal tissues such as stomach and salivary glands (9, 23, 44). However, in contrast to iodine, 99mTcO4 is not incorporated into thyroglobulin after being trapped, and leaves thyroid follicular cells within hours (3, 38).
Although data for 99mTcO4 thyroid uptake are well-established as scintigraphic parameters for thyroid gland functional status in humans with both normal gland function and a variety of thyroid diseases (35), there are few data available in mice for research study purposes. As the primary regulator of body metabolism, thyroid hormone is a key component of physiological homeostasis in humans and animals. Normal and disease conditions affect the thyroid in feedback and response loops, and body tissue thyroid hormone levels influence stress responses. The critical role that pre-clinical animal models of disease play in understanding disease mechanisms make thyroid function status an important consideration for research study design in these models. There are few published references on thyroid function and uptake in mice. One of the main reasons is that spatial resolution in conventional clinical gamma cameras, even when equipped with pinhole collimators, is low and of limited use for thyroid scintigraphy in mice (31, 36). High spatial resolution is required for studying small foci such as the mouse thyroid gland which can be as small as 1 mm3 (6). In addition, the thyroid and the salivary glands (submandibular-sublingual salivary gland complex) in mice are in close proximity in the mouse neck region (▶ Fig. 1). This anatomic proximity and the fact that both organs accumulate NIS substrates such as 99mTcO4 make it difficult to distinguish these organs from each other and to quantify their uptake separately in planar or single-photon emission computed tomography (SPECT) conventional imaging (10, 18). Therefore, small-animal studies for thyroid function have relied on
Fig. 1 In vivo SPECT/CT images of the head-neck region of a 6-month-old female mouse showing normal thyroid uptake 30 min after intravenous injection of 65 MBq 99mTcO4, in transverse (A), coronal (B) and sagittal (C) projections fused with CT images. Uptake foci are located in the thyroid (T), the submandibular-sublingual salivary gland complex (SSC), and parotid glands (PG).
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assays of the specific radioactivity content in thyroid tissue after euthanasia. Recent developments in high-resolution multi-modality imaging devices such as small-animal SPECT/CT for dedicated rodent imaging offer improved organ discrimination and uptake determination accuracy. These new scanners can achieve gamma emitting radioisotope images in small objects with less than 1-mm spatial resolution (24, 26) which is sufficient for studying the thyroid gland in small animals. Imaging approaches also allow examination of the same mouse non-invasively, and repeatedly in longitudinal studies, facilitating the use of animals as their own comparison controls (24, 25, 28). Despite the introduction of high-resolution SPECT/CT systems for small-animal imaging, however, there are no established procedures for thyroid scintigraphy in mice. The aims of the present study were to develop a protocol for small-animal SPECT thyroid imaging and to compile normal 99mTcO thyroid uptake values as reference 4 for future studies using standard thyroid scintigraphy with 99mTcO4. A further goal was to identify and systematically investigate potential physiological factors that might have a significant influence on 99mTcO thyroid uptake, i.e. age and sex of 4 the animal, and the time of day as a result of animal circadian rhythm.
Materials and Methods Animals and study design The study protocol was approved by the local Committee for Animal Care (reference number G0353/12) according to the German Law for the Protection of Animals. All applicable institutional and national guidelines for the care and use of animals were followed. Twelve healthy female and 12 healthy male C57BL/6N mice were bred at the Charité – Universitätsmedizin Berlin, Germany, and were brought to the animal husbandry unit at the Berlin Experimental Radionuclide Imaging Center (BERIC) at time of weaning (21 days). Animals were housed under standardized conditions in a climatic chamber (Bioscape, UniProtect, © Georg Thieme Verlag KG 2018
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Germany; temperature 22 ± 2 °C, relative humidity 50 ± 5 %), and a light-dark-cycle following a 12-hour rhythm with light conditions from 7 am to 7 pm and dark conditions from 7 pm to 7 am was applied. Animals lived in four groups of six animals each and were separated by sex. Mice were kept in filter topped Makrolon type III cages (37 x 21 x 18 cm) with sawdust bedding and environmental enrichment such as tissue material for nest building, one red plastic nest box, a plastic tunnel and a wood stick for gnawing. Animals received both a standard diet (SSNIFF®, Soest, Germany) and water ad libitum. Each mouse was imaged in longitudinal follow-up studies up to an age of 24 months. After an adaptation period of at least 7 days, the first examination was performed at a pubertal age of approximately 1 month (median age 1.1 months, range 0.9 – 1.2; body weight 16.9 g, 13.8 – 21.7). At the time of the second examination mice were 3 months old, sexually mature, but not fully grown (age 2.9 months, 2.4 – 3.9; body weight 23.6 g, 19.8 – 32.8). The third examination was performed at an age of 6 months, when mice were fully grown (age 5.8 months, 5.5 – 7.3; body weight 31.2 g, 22.9 – 41.3). Thereafter, mice were examined at 12 months (age 12.1 months, 11.8 – 13.1; body weight 36.2 g, 23.8 – 48.4) and 24 months of age (senile, age 23.6 months, 22.9 – 24.1; body weight 37.9 g, 27.4 – 45.9). In order to monitor circadian rhythm, imaging was performed during the first half of light conditions during the main
hours of the rest-and-sleep phase as well as during the first half of night conditions in the main phase of locomotor activity. To minimize physical stress in juvenile onemonth-old animals, only one examination was performed, either during light or during night conditions. To exclude potential effects of anesthesia on circadian rhythm, an interval of at least 84 hours between examinations was granted to each animal. Additionally, to avoid potential bias by carry-over effects from the preceding anesthesia, half of each group was studied under daytime light conditions first and subsequently under night conditions, whereas examinations in the other group half began with initial studies at night. During aging, some animals were excluded from the study because of health problems. As a result, the initial number of 12 female and 12 male mice decreased during the study over 24 months. Missing animals were not replaced in this longitudinal study, because mice served as their own study control comparison for multiple observations. Details regarding the number of animals at each study time-point are shown in ▶ Table 1.
SPECT/CT imaging SPECT and CT imaging were performed using the NanoSPECT/CTplus scanner (Mediso, Hungary /Bioscan, France). The dual modality system for dedicated small animal imaging is based on a four-head gamma camera with NaI(TI) crystal detectors. Each detector was equipped with a
nine-pinhole aperture (mouse high resolution, d = 1.0 mm) with a spatial resolution of 0.7 mm FWHM for 99mtechnetium (26). SPECT images were acquired using 20 projections consisting of five steps displaced by 18° and the energy window was set at 140 keV ± 10 %. Mice were first weighed and then anesthetized using 1–2 % isoflurane with oxygen at a flow rate of approximately 0.5 l / min. Accounting for circadian rhythm, anaesthetized mice were injected into the tail vein with approximately 80 MBq of 99mTcO4 (0.1 – 0.15 ml) either from 7.30 am to 2.40 pm or 4.30 pm to 11.10 pm. The equipment used for intravenous injection was constructed from a 30 G cannula and a 0.28 x 0.61 mm catheter (A. Hartenstein, Portex, Germany). Immediately after injection, mice were immobilized in prone position on a heated (37 °C) mouse bed (Equipement Vétérinaire Minerve, France) and kept anesthetized during the imaging protocol with isoflurane. A respiratory pillow was used to monitor respiration frequency (70–80 / min) for anesthesia adjustment throughout image acquisition. After a low-dose CT scout scan for positioning the thyroid region in the 20 mm field of view, nine consecutive multi-pinhole SPECT images of 10 min duration each (5 angular steps at 60 sec each, 2 bed positions) were acquired starting approximately 5 min after intravenous injection, followed by a CT scan. Because the rotation time of the gantry adds to the scan duration, the total SPECT imaging time
Tab. 1 99mTcO4 thyroid uptake (%ID) with respect to age, sex and circadian rhythm. Mice were imaged in longitudinal follow-up studies up to an age of 24 months. The sample size of 6 animals with an age of 1 month is lower than in all other groups, because juvenile mice were imaged either only during sleep or awake phase, whereas at all the other time points, mice were imaged during both sleep and awake phases. Each set of data includes the median (%ID), interquartile range [IQR], min-max at time of maximum uptake within the 97-minute SPECT acquisition and number of animals. Sex
Age(months)
1 month
3 months
6 months
12 month
24 months
sleep
awake
sleep
awake
sleep
awake
sleep
awake
sleep
awake
females
median [IQR] min – max no. of animals
1.6 [1.5–1.7] 0.9–2.1 6
1.6 [1.4–1.6] 1.4–1.7 6
1.5 [1.4–1.7] 1.2–1.9 11
1.8 [1.7–1.9] 1.4–2.0 11
1.8 [1.7–2.0] 1.5–2.3 10
1.8 [1.7–2.0] 1.1–2.2 10
1.6 [1.6–1.7] 1.4–1.7 10
1.5 [1.4–1.7] 1.2–1.9 10
1.1 [1.0–1.3] 0.8–1.7 4
1.1 [1.1–1.2] 1.0–1.5 8
males
median [IQR] min – max no. of animals
1.1 [1.0–1.2] 0.9–1.3 6
1.2 [1.1–1.2] 1.0–1.3 6
1.2 [1.1–1.2] 0.9–1.4 11
1.1 [0.9–1.1] 0.8–1.3 11
1.2 [1.1–1.3] 1.0–1.4 11
1.1 [1.0–1.2] 0.9–1.5 10
1.2 [1.1–1.3] 1.0–1.3 10
1.1 [1.0–1.2] 0.8–1.3 10
1.2 [1.1–1.3] 0.7–1.7 4
1.1 [1.0–1.2] 0.9–1.3 8
© Georg Thieme Verlag KG 2018
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Beindorff N et al.: Thyroid uptake of 99mTc-pertechnetate SPECT in mice
summed to 97.47 min based on an actual scan duration of 10.83 min per image. After scanning, the mice were put into a chamber (Peco Services, MediHeat, UK; 35°C) for anesthesia recovery. In 6 female and 6 male mice at ages of 1 and 6 months, an additional scan of 20 min duration (5 angular steps at 120 sec each, 2 bed positions) was performed 5 to 9 hours after injection to assess thyroid 99mTcO4 retention.
SPECT image reconstruction and quantification SPECT images were reconstructed using an ordered subset expectation maximization (OSEM) algorithm (HiSPECT, Bioscan, Washington, DC, USA) with standard parameter settings (3 x 3 iterations; 35 % smoothing). Thyroid gland 99mTcO4 uptake was determined by manual contouring of a volume-of-interest (VOI) using PMOD 3.4 (PMOD Technologies Ltd., Switzerland) as shown in ▶ Fig. 2. The VOI for the dynamic image data set was drawn in the time frame with the highest thyroid uptake allowing separation of thyroid uptake from that of salivary glands on the transverse projections used for VOI definition. An isocontour at 5 % of maximum thyroid activity was generated to reduce spill-over from the adjacent salivary glands and applied to the other dynamic frames. The time activity curve (TAC) obtained was corrected for radioactive decay, adjusted for the elapsed time between injection and start of scanning. The percentage tracer uptake in the thyroid was calculated by normalizing the integrated activity of each
Fig. 2 99mTcO4 uptake in the thyroid gland was determined in the transverse projection by manual contouring of a volume-of-interest (VOI).
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VOI at the time of maximum uptake to the total injected activity (%ID). CT images did not allow visual separation of thyroid gland tissue from surrounding soft tissue. Therefore, SPECT images were used as a substitute for thyroid volume assessment taking into account that SPECT does not yield data on organ absolute size and volume. The SPECT VOI for thyroid was defined using PMOD and contouring the 5 % isocontour outline in the time frame with highest uptake. To separate parts of the salivary glands for image analysis, the VOI isocontours were corrected manually when necessary.
Statistical analysis Statistical analysis for study data was performed with R 3.4.1. (The R Foundation for Statistical Computing, Vienna, Austria). Due to the small study sample size, nonparametric data distribution was assumed. Metric data are presented as median, interquartile range [IQR, 25th-75th percentile], minimum and maximum and visualised as boxplots. Paired data with more than two groups were analysed using the Friedman test with Bonferroni-Holm adjustment for pairwise post-hoc Wilcoxon tests. Comparison of unpaired groups was performed using the Mann-Whitney-U test. All tests were two-sided and significance of differences was assumed at p < 0.05. Association of two metric parameters was analyzed using Spearman‘s rho correlation coefficient for non-parametric data.
Thyroid 99mTcO4 uptake kinetics
▶ Fig. 3 shows typical thyroid uptake in
mice during the first 100 min after 99mTcO4 injection and after 6 – 8 hours. A steep increase in thyroid 99mTcO4 uptake occurred after tracer injection with the maximum uptake reached between 8 and 45 min (median 26.9 [22.2 – 31.4] 8.4 – 43.3). The subsequent excretion rate was slower. The T75 (25 % clearance) was 75.8 min ([65.7 – 85.9] 37.5 – 97.6). T50 (clearance half time) was 138.5 min ([125.6 – 174.9] 100.3 – 215.1) and could only be calculated in animals which were scanned again 5 – 9 hours after injection. At the end of the imaging period (5 – 9 hours) 99mTcO4 thyroid uptake was still visible. At an age of 1 month female mice revealed a significant difference in time-topeak thyroid uptake throughout the day (sleep 30.6 min [27.3 – 33.3] 23.1 – 40.5; awake 21.1 min [19.3 – 24.6] 14.7 – 27.7; p = 0.015) compared to males (sleep 25.9 min [21.8 – 28.3] 20.4 – 28.9; awake 15.7 min [9.7 – 21.2] 9.4 – 29.8; p = 0.2). Both sexes showed no significant circadian rhythm related variations on time-to-peak thyroid uptake. With aging, a tendency (p = 0.059) towards a delayed time-to-peak uptake was observed in male mice (▶ Fig. 4). Within age groups, no significant differences in time-to-peak thyroid
Results According to the study protocol, the mouse head-and-neck region was imaged after intravenous injection of 99mTcO4. The locations of the thyroid and salivary glands are illustrated in ▶ Fig. 1. As shown in ▶ Fig. 1A, the transverse projection was best suited for distinguishing between the thyroid, the submandibular-sublingual salivary gland complex and parotid glands. These images demonstrate that dedicated small animal imaging devices can be used to depict the two independent thyroid lobes in rodents.
Fig. 3 Thyroid uptake time-activity-curves from 9 consecutive multi-pinhole SPECT images of 10 min duration each, starting at 5 min after intravenous injection of 70 MBq 99mTcO4 in two 1-month-old female mice. Thyroid uptake reached a maximum in mouse 1 at approximately 22 min post injection and in mouse 6 approximately 31 min after injection. Thyroid uptake decreased slowly over time in both animals and remained measurable up to 8 hours after injection.
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Fig. 5 99mTcO4 mouse thyroid uptake (%ID) with respect to age and sex. Female mice had a significantly higher thyroid uptake than males up to age 12 months and showed uptake as an inhomogeneous population parameter with respect to age (p = 0.056). The uptake in males was a homogeneous population parameter in all age groups (p = 0.267).
uptake were observed between males and females. In general, there was no significant difference between females (26.7 min [23.0 – 30.8] 8.4 – 43.3) and males (27.0 min [21.6 – 31.9] 8.4 – 42.2; p = 1) in the time to peak thyroid uptake. However, females (73.2 min [64.1 – 82.5] 37.5 – 97.5) showed a tendency to an earlier T75 compared to males (78.6 min [70.7 – 87.1] 42.7 – 97.6; p = 0.06).
Thyroid uptake (%ID) Thyroid uptake (%ID) as used in this manuscript refers to the maximal uptake at the time of maximum uptake (tmax) within the 97-minute SPECT acquisition. C57BL/6N mice showed a maximum 99mTcO thyroid uptake of 1.3 %ID 4 ([1.1 – 1.6] 0.7 – 2.3) between 8 and 45 min after injection of 99mTcO4. The six female and six male mice that were imaged additionally 5-9 h (median 6.3 h) after injection had a remaining uptake of 0.2 %ID ([0.1 – 0.3] 0.1 – 0.5) which corresponded to 16.1 % ([13.9 – 19.6] 6.9 – 27.9) of the maximum uptake. A summary of the maximum © Georg Thieme Verlag KG 2018
thyroid uptake values is shown in ▶Table 1. Circadian rhythm: No significant effects of circadian rhythm were observed in females (sleep 1.6 %ID [1.5 – 1.7] 0.8 – 2.3; awake 1.7 %ID [1.4 – 1.8] 1.0 – 2.2; p = 0.65). The difference in uptake between sleep and awake phase in male mice achieves borderline significance (sleep 1.2 %ID [1.1 – 1.3] 0.7 – 1.7; awake 1.1 %ID [1.0 – 1.2] 0.8 – 1.5; p = 0.02). Age: The relationships between animal sex and age are shown in ▶ Fig. 5. A sex-related influence of age on thyroid uptake is observed in female mice, because they show variable thyroid uptake with respect to age (p = 0.056). Initially, females showed a non-significant increase in median thyroid uptake up to age 6 months. Thereafter, a significant decline occurred at age 12 months (p = 0.02) and at age 24 months (p = 0.048). In contrast, thyroid uptake in males was uniform with aging (p = 0.27). Sex: ▶ Fig. 5 and ▶ Table 1 illustrate that female mice had a significantly higher thyroid uptake than males up to age 12 months (12 months: females 1.6 %ID [1.5 – 1.7] 1.2 – 1.9, males 1.2 %ID [1.1 – 1.2]
0.8 – 1.3; p < 0.001). Thereafter, thyroid uptake in female mice declined to the level of males (24 months: females 1.1 %ID [1.1 – 1.3] 0.8 – 1.7, males 1.1 %ID [1.1 – 1.2] 0.7 – 1.7; p = 0.93). In contrast, male animals showed no change in thyroid uptake up to an age of 24 months. While uptake values for male mice show less variability, the values for individual female mice varied to a greater extent (females 1.6 %ID [1.5 – 1.8] 0.8 – 2.3, males 1.1 %ID [1.1 – 1.2] 0.7 – 1.7; p < 0.001). SPECT images were used for approximate volume determinations of the thyroid gland. Between ages of 1 and 3 months, both female (11.8 mm3 [11.4 – 12.6] 10.3 – 14.0 and 12.8 mm3 [12.0 – 13.8] 10.2 – 14.9, respectively; p = 0.04) and male mice (11.1 mm3 [10.9 – 12.4] 10.4 – 13.4 and 13.1 mm3 [12.7 – 14.1] 11.8 – 16.7, respectively; p < 0.01) had significant thyroid growth. At later time-points, female mice had no significant changes in thyroid volume (24 months: 12.9 mm3 [11.7 – 13.9] 11.0 – 14.7; p = 0.8). In contrast, the thyroid volume in male mice increased significantly between 3 and 12 months (12 months: 14.8 mm3 [14.2 – 15.6] 11.9 – 18.3; Nuklearmedizin 5/2018
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Fig. 4 Time-to-peak (tmax) of 99mTcO4 thyroid uptake in minutes with respect to mouse age and sex. With aging, a tendency towards a delayed time-to-peak uptake was observed in male mice (p = 0.059).
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p = 0.046). Overall, males (13.9 mm3 [12.7 – 15.2] 10.4 – 21.0) showed a larger thyroid gland volume compared to females (12.7 mm3 [11.8 – 13.5] 10.2 – 14.9; p < 0.001). As female mice had generally higher total thyroid uptake and smaller gland volume, female mice had a higher uptake per mm3 of thyroid gland tissue (female 0.13 %ID / mm3 [0.12 – 0.14] 0.05 – 0.18; male 0.08 %ID / mm3 [0.07 – 0.09] 0.04 – 0.13; p < 0.001). After normalizing the thyroid volume to body weight females (0.51 mm3 / g [0.40 – 0.62] 0.29 – 0.99) had a significantly larger relative thyroid volume than males (0.45 mm3 / g [0.39 – 0.50] 0.29 – 0.75; p < 0.01). With aging, thyroid volume did not correlate significantly with body weight in females (rho = 0.15; p = 0.17), however, in males a correlation was found (rho = 0.59; p < 0.001).
Discussion Murine models represent the most flexible and commonly used models for pre-clinical investigations for thyroid disease, but also other diseases and therapeutic approaches with impact on thyroid function. In recent years, the extensive development and implementation of murine gene knock-out and knock-in models have become especially powerful systems for investigations into disease mechanisms. Like humans, animals have physiological variations resulting from changes in serum and tissue hormone levels. These changes can have significant effects on metabolism, disease development and behavior. The thyroid hormone axis and its effects on overall metabolism and the significant interactions with other major hormone axes are key factors in animal model physiology. A complete characterization of a pre-clinical murine model should include an understanding of the possible effects of differences in thyroid function in animals of different sexes and ages to assure an objective interpretation of study results. The present study established normal 99mTcO thyroid uptake values in healthy 4 C57BL/6N mice over a time period of 24 months. This was necessary because there are little published data on murine thyroid Nuklearmedizin 5/2018
uptake. Furthermore, there are no publications on the effects of age, sex and circadian rhythm on thyroid 99mTcO4 uptake in mice. In this longitudinal study, scintigraphic findings were not supported with animal hormone status determination (serum thyroid hormones, estrogens and progesterone levels, etc.). An average 25 g mouse has a small total blood volume of approximately 1.7 ml. This small blood volume is not sufficient for frequent sampling and preservation of a healthy animal. The present study also presents a standardized, reproducible SPECT imaging procedure for thyroid 99mTcO4 uptake assessment. Because maximum thyroid uptake in mice occurs between 8 and 45 minutes, image acquisition over a period of 60 min with six consecutive images of 10 min duration each after injection of 99mTcO is recommended. 99mTcO injection 4 4 can be performed before positioning the animal in the SPECT/CT scanner. This protocol will allow reliable determination of the thyroid uptake maximum irrespective of animal age, sex and circadian rhythm. The mice in this study had a maximum 99mTcO thyroid uptake of 1.3 %ID 4 ([1.1 – 1.6] 0.7 – 2.3) which is similar to other studies in mice with 1.9 %ID ± 0.6 (18) as well as clinically euthyroid humans with 99mTcO4 uptakes ranging from 0.4 to 1.7 %ID (35). It should be pointed out, however, that all these data might be murine strain specific and, thus, only valid in C57BL/6N mice.
Circadian rhythm Most animal experiments are performed during daytime and/or the evening across the main phases of both resting and sleeping and awake phase of nocturnal animals like mice (40). This schedule accommodates laboratory working hours, but does not account for the physiological circadian rhythms of rodents. Numerous physiological functions in humans and mice are affected by the circadian rhythm. Examples are locomotor activity phases, sleep-awake cycles, blood pressure regulation, core temperature, heart rate rhythm, and endocrine functions such as the hypothalamic-pitu-
itary-thyroid rhythm (2, 32, 42). Mice show diurnal changes in locomotor activity and core temperature with maxima in dark hours and secondary maxima of about one hour duration immediately following the beginning of light time (42). The circadian system exerts control at all levels of the hypothalamic-pituitary-thyroid axis. The chronobiology in the endocrine system (21) shows that the hypothalamic content of thyroid-releasing hormone (TRH) follows a circadian rhythm in rodents, which is light-dark cycle-dependent. TRH is the main stimulator of thyroid-stimulating hormone (TSH) production and release and, therefore, appears to be the most important factor in the regulation of the pulsatile secretion and of TSH circadian rhythm. In male mice, statistically significant differences in 99mTcO4 uptake between sleep and awake phases were observed, which is compatible with the circadian rhythm of TSH secretion. Because these differences in uptake of approximately 0.1 %ID, less than 10 % of total uptake, are small, they are likely of limited relevance for the interpretation of imaging study data in mice. Interestingly, no significant circadian differences of 99mTcO4 uptake were observed in female mice. As uptake values for female mice vary to a greater extent in similar age groups, small circadian rhythm related differences observed in male mice probably go undetected in females due to their greater variation in thyroid 99mTcO4 uptake values. From these results, it cannot be concluded that circadian rhythm related effects exist in female mice, although small circadian rhythm related effects seem likely in analogy to the observed effects in male mice.
Age Male mice showed stable thyroid 99mTcO4 uptake values over the entire period of investigation up to 24 months. Female mice showed a significantly higher thyroid uptake than males which declined between ages of 12 and 24 months, finally reaching the level of males at 24 months. This decline correlates with murine reproductive senescence transition. Mice are sexually mature by 3 – 6 months of age (14). In © Georg Thieme Verlag KG 2018
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C57BL/6J female mice, dysfunction of luteinizing hormone (LH) secretion and glial hyperactivity are concurrent with the lengthening and loss of reproductive hormonal cycles with persistent anestrus (10 – 14 months) (16). In this study, radiation effects from repeated injections of 80 MBq 99mTcO4 on the thyroid appeared to be negligible, because male mice showed stable thyroid 99mTcO4 uptake over the entire study period, while uptake in female mice correlated with ovarian activity changes during aging. A possible thyroid uptake stunning effect from 99mTcO administration for up to two days 4 as reported by Cambien et al. (12) can be excluded in results interpretation, because 99mTcO injections were administered at a 4 minimum interval of 84 h. The thyroid volume of an adult C57BL/6 mouse measured by ultrasound ranges between 2.1 and 4.9 µl (29). Because of the object blurring effects (partial volume effect and spill-over) in planar images, SPECT images were used to make thyroid volume estimates. In the present study, thyroid volumes were likely overestimated. Being aware of the fact that SPECT is not a reliable tool for absolute size measurements, relative size of the thyroid was assessed during animal aging. Female mice had an increase in thyroid volume up to an age of 3 months with little change in volume thereafter. In contrast, male mice had continuous thyroid growth up to an age of 12 months. The findings of male thyroid growth are supported by older publications reporting an increase in mouse thyroid size and histological changes such as increasing interstitial fibrosis with age appearing at about 17 months (8). The histological change with age in both animal sexes consisted of central follicle enlargement with gradual atrophy of the lining epithelium, including fibrous tissue replacement. Epithelial atrophy could be an explanation for the decrease in thyroid 99mTcO4 uptake in female mice observed after 12 months of age while this effect could be overcome by the increase in size in males resulting in stable thyroid uptake values.
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Sex The study results show that female mice have significantly higher thyroid 99mTcO4 uptake than males up to an age of 12 months. Thereafter, with reproductive senescence, the 99mTcO4 thyroid uptake in female mice declined to the level of males. As female mice had generally a higher total thyroid uptake and smaller thyroid volume, female mice had a higher uptake per mm3 of thyroid gland tissue, compared to males. However, uptake values for female mice varied to a greater extent in similar age groups. These results suggest that 99mTcO4 thyroid uptake in female mice is influenced by the estrous cycle. In C57BL/6J mice, the estrous cycle is divided into 4 stages (proestrus, estrus, metestrus, and diestrus) and repeats every 4 to 5 days (1, 11). Serum estradiol (E2) and progesterone (P4) levels in C57BL/6 mice vary throughout the cycle (43). The frequency of estrous cycles begins to decline at about 9 months of age (33) and the age of onset of acyclicity ranges between 11 to 16 months (15). Follicular reserves are nearly exhausted at the cessation of cyclicity (20). Plasma E2 concentration falls and LH concentration rises to levels observed in ovariectomized mice after the cessation of persistent vaginal cornification in C57BL/6J mice (19). These physiological changes in the estrous cycle with age parallel the observed decrease in 99mTcO4 thyroid uptake in female mice after the age of 12 months. Ayala et al. showed that circulating TSH levels in euthyroid rats change during the estrous cycle with highest levels occurring during proestrus and lowest levels during estrus and diestrus (4). Both NIS and the multifunctional anion exchanger pendrin (SLC26A4/PDS) expression and activity are increased by TSH (7). The interdependency of the thyroid and ovarian hormones is apparent. Estrogens may regulate the expression of genes that codify essential proteins for hormonal biosynthesis such as thyroperoxidase (TPO) and NIS (27). Our data support the influence of estrogens in female mice on 99mTcO4 thyroid uptake. In contrast, male mice in this study showed no changes in thyroid uptake up to an age of 24 months. Uptake values in individual
male animals also showed less variability compared to females. Other investigators found that plasma testosterone levels remained almost constant during the average lifespan of male C57BL/6 mice (17). This explains the stable uptake values in male mice in all age groups as observed in our study. In addition to providing a protocol for the use of 99mTcO4 uptake for murine thyroid function research, this study provides critical insight on parameters affecting 99mTcO uptake for further use in murine 4 model investigations for other basic and disease research. Pre-clinical murine disease models serve an important function in assessing treatment regimens, target effects and therapeutic efficacy. Animal thyroid function is the primary control for animal physiological functions, and most significantly, for overall metabolism. Planning and interpretation of studies in murine pre-clinical models needs to include considerations of the thyroid function and its control by physiological influences from sex, estrous status, age and circadian rhythm as these influences can have significant effects on animal responses to experimental variables. These influences should be also considered in other murine model research areas involving thyroid transporter function imaging such as in cell NIS gene transfection. SPECT tumor imaging has been performed in murine models after NIS gene transfection (13) and 131iodine therapy studies (13, 37, 39). NIS cell transfection is also used to create a reporter gene for monitoring transfected cells, e.g. for stem cell biokinetic studies and their target organ homing (41). A PET tracer for NIS imaging is the new agent 18F-tetrafluroborate, which has biodistribution characteristics similar to 99mTcO4. It is taken up selectively in NIS-expressing tissues in both mice and humans. This tracer for PET imaging is possibly a promising surrogate for 99mTcO SPECT (22, 30, 34). 4
Conclusion The present study demonstrates a significant influence of animal sex on 99mTcO4 thyroid uptake in mice. Female mice had Nuklearmedizin 5/2018
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Beindorff N et al.: Thyroid uptake of 99mTc-pertechnetate SPECT in mice
Beindorff N et al.: Thyroid uptake of 99mTc-pertechnetate SPECT in mice
significantly higher thyroid uptake maxima as well as higher uptake variability compared to males. These results suggest that 99mTcO thyroid uptake in female mice is 4 influenced by the estrous cycle of the animal. Sex is also a significant factor for agerelated changes, which were observed in female but not in male animals, also likely triggered by influences from the estrous cycle. In contrast, circadian rhythm had no significant impact on thyroid 99mTcO4 uptake in female mice while in males, the effects were minimal. The design of future studies on 99mTcO4 thyroid uptake in mice therefore should consider sex and, in female study groups, age as key factors that impact on study results. Acknowledgements
This work was supported in part by the Technologiestiftung Berlin (TSB) for SPECT/CT use. This manuscript is based partially on the results of the doctoral thesis of Annika Bartelheimer. We are very thankful for the excellent technical assistance of Betina Gregor-Mamoudou. Conflict of Interest
The authors declare that they have no conflict of interest.
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