PET quantification of [18F]MPPF in the canine brain using blood input and reference tissue modelling

Numerous studies have shown that the serotonin1A (5-HT1A) receptor is implicated in the pathophysiology and treatment of several psychiatric and neurological disorders. Furthermore, functional imaging studies in a variety of species have demonstrated that 4-(2´-Methoxyphenyl)-1-[2´-(N-2´´-pyridinyl)-p- [18F]fluorobenzamidoethylpiperazine ([18F]MPPF) is a valid and useful PET tracer to visualize the 5HT1A receptor. However, to our knowledge, [18F]MPPF has never been demonstrated in the canine brain. The ability to image the 5HT1A receptor with PET in dogs could improve diagnosis and therapy in both canine and human behavioural and neuropsychiatric disorders. To examine the potential use of [18F]MPPF in dogs, five healthy adult laboratory beagles underwent a 60-minutes dynamic PET scan with [18F]MPPF while arterial blood samples were taken. For each region of interest, total distribution volume (VT) and corresponding binding potential (BPND) were calculated using the 1-tissue compartment model (1-TC), 2-Tissue compartment model (2-TC) and Logan plot. The preferred model was chosen based on the goodness-of-fit, calculated with the Akaike information criterium (AIC). Subsequently, the BPND values of the preferred compartment model were compared with the estimated BPND values using three reference tissue models (RTMs): the 2-step simplified reference tissue model (SRTM2), the 2-parameter multilinear reference tissue model (MRTM2) and the Logan reference tissue model. According to the lower AIC values of the 2-TC model compared to the 1-TC in all ROIs, the 2-TC model showed a better fit. Calculating BPND using reference tissue modelling demonstrated high correlation with the BPND obtained by metabolite corrected plasma input 2-TC. This first-in-dog study indicates the results of a bolus injection with [18F]MPPF in dogs are consistent with the observations presented in the literature for other animal species and humans. Furthermore, for future experiments, compartmental modelling using invasive blood sampling could be replaced by RTMs, using the cerebellum as reference region.


Introduction
The serotonin 1A (5-HT 1A ) receptor is a G-protein-coupled receptor and is believed to be one of the most important 5-HT receptor subtypes [1][2][3]. Two different 5-HT 1A receptor populations can be found in the mammalian brain [4][5][6][7][8]. The first population is located in the raphe nucleus and has an auto-receptor function that inhibits serotonin release at the nerve terminals. The second population is highly abundant in cortico-limbic areas, were it acts as postsynaptic receptor [2,9].
The 5-HT 1A receptor is involved in the pathophysiology of a variety of psychiatric and neurological disorders including depression, anxiety, schizophrenia, dementia, eating disorders, hallucinogenic behaviour, epilepsy and motion sickness [6,[10][11][12][13][14]. Functional imaging studies of this receptor in healthy volunteers and in people suffering from neuropsychiatric disorders could improve diagnosis and therapy of these diseases. Therefore, several radioligands have been studied to assess in vivo changes in the 5HT 1A receptors using positron emission tomography (PET). Several PET-studies have demonstrated that 4-(2´-Methoxyphenyl)-1-[2´-(N-2´´-pyridinyl)-p-[ 18 F]fluorobenzamidoethylpiperazine ([ 18 F]MPPF) is a valid and reliable PET-tracer for imaging the 5HT 1A receptor in the human brain [10,15]. In vitro binding assays with the tritiated compound showed that this 5-HT 1A antagonist has a high affinity (Kd: 0.34 nM) and good selectivity towards the 5-HT 1A receptors [16]. Other advantages of this radiotracer are a high initial brain uptake, the relatively simple radiochemical synthesis compared to other 5HT 1A PET-tracers (for example: [ 11 C]WAY100635) and the long physical half-life of 18 F, which permits distribution from production facilities to remote hospitals [1,10,17]. To date, [ 18 F]MPPF has been used successfully in rodents [18][19][20][21], cats [7,22,23], non-human primates [24,25] and humans [10,15,[26][27][28]. Unfortunately, to the authors' knowledge, it has never been used in dogs. However, functional imaging of the 5-HT 1A receptor in dogs could offer several opportunities as dogs exhibit naturally occurring behavioral disorders that may be comparable to certain human psychiatric conditions [29][30][31][32][33]. Therefore, dogs are considered as a valuable animal model for several human psychiatric disorders. Moreover, as opposed to rodents, dogs have a substantial amount of frontal cortex [34], which makes them a more interesting and more practical animal model in human psychiatry. Radioprotective regulations are also less stringent in dogs than in humans, allowing longitudinal studies with repeated PET scans in a relative short time frame. In addition, functional imaging studies of the 5-HT 1A receptor in dogs could also improve our understanding of several canine behavioral disorders and may serve as a tool to improve diagnosis and treatment of these disorders.
The objective of this study was to quantify dynamic [ 18 F]MPPF PET data, obtained in healthy beagle dogs, in pharmacological parameters utilizing compartmental modeling. Several standard kinetic models were evaluated using metabolite corrected arterial plasma input functions and time-activity curves of each region of interest. Subsequently, to avoid invasive arterial blood sampling in future experiments and to improve the clinical applicability of the analysis method, the validity of different reference tissue models was assessed.

Experimental animals
This study was approved by the Ethical committee of Ghent university (EC approval 17/108). Five neutered female experimental beagles (age 4.7 ± 0.1 years; weight 11.2 ± 0.7 kg) were included in this study. The animals were classified as healthy based on general clinical examination. They were provided by the faculty of veterinary medicine (Ghent University) and were socially-housed in small groups (2 to 8 dogs) on an internal surface of 15 m 2 with permanent access to an outside area of 15 m 2 , equipped with enrichment tools. Food was withheld for at least 12 hours before anaesthesia but water was provided ad libitum. Prior to transportation to the PET centre of Ghent university hospital, the dogs were premedicated with an intramuscular (i.m.) injection of dexmedetomidine (375 μg/m 2 body surface area, Dexdomitor, Orion Corporation, Espoo, Finland). At the PET centre, a 22G over-the-needle venous catheter was placed in one of the cephalic veins to gain intravenous access and a Lactated Ringer's solution (Vetivex 500 mL, Dechra Veterinary products, Heusden-Zolder, Belgium) was infused i.v. at a rate of 5 mL/kg/h. General anaesthesia was induced with propofol (Propovet, Abbott laboratories, Queenborough, UK) given i.v. to effect. During the scan, anaesthesia was maintained with a mixture of 1.2-1.4% isoflurane (Isoflo, Abbott laboratories, Queenborough, UK) vaporized in oxygen using a circle rebreathing system. After positioning the dog on the bed of the PET/ CT scanner, a 22G arterial catheter was placed in one of the dorsalis pedis arteries to perform arterial blood sampling. During general anaesthesia, a continuous monitoring of body temperature and cardiorespiratory functions by pulse oximeter and capnography was performed. After completion of the study, the dogs were reintroduced in their housing facility and remained available for future research.

Image analysis
Prior to the PET scan, a series of T1 weighted anatomical images (3D MPRAGE sequence, 176 sagittal slices, TR: 2250 ms, TE: 4.18 ms, TI: 900 ms, parallel acquisition method = GRAPPA acceleration factor: 2, matrix size: 256 x 256, FOV: 220 mm, flip angle: 8˚, voxel size: 1 x 1 x 1 mm 3 ) were acquired using a 3T Magnetom Trio Tim system MRI scanner (Siemens, Erlangen, Germany). Subsequently, these images were co-registered with the PET/CT images using PMOD software version 3.405 (PMOD Technologies, Ltd., Zurich, Switzerland). Based on information from a dog brain atlas [35], 16 regions of interest (ROI) were manually delineated on the MR image. These included: left frontal cortex, right frontal cortex, left temporal cortex, right temporal cortex, left occipital cortex, right occipital cortex, left parietal cortex, right parietal cortex, midbrain, anterior cingulate gyrus, posterior cingulate gyrus, left hippocampus, right hippocampus, subgenual cingulate gyrus, presubgenual cingulate gyrus and cerebellum. Subsequently, time-activity curves could be obtained for each ROI representing the radioactivity concentration into each time frame, corrected for decay.
For each of the dogs, a Watabe function was fitted to the six metabolite time points and added to the plasma input function to obtain a metabolite-corrected plasma input function [36].

Kinetic modelling
All kinetic modelling was performed using the kinetic tool of the PMOD software version 3.405. The total volume of distribution (V T ), defined as the ratio of the concentration of radiotracer in a certain ROI to the concentration in plasma at equilibrium, was calculated for the different ROIs using the one-and two-tissue compartment (1-TC and 2-TC) model [37] and the Logan plot [38].
In order to validate that a reference tissue model constitutes as a good alternative for the invasive blood sampling, the non-displaceable binding potential (BP ND ), referring to the ratio at equilibrium of specifically bound radioligand to that of non-displaceable (ND) radioligand in tissue, was estimated for both compartment models and the Logan plot [39]. Therefore, a region devoid of the targeted receptors (i.e. a reference region) is required. Various in vitro and in vivo experiments have demonstrated the absence of 5HT 1A receptors in the cerebellum, and therefore, the cerebellum has been put forward as reference region [19,[39][40][41].
The goodness-of-fit for each compartment model was evaluated using the Akaike Information Criterion (AIC) value, where the lower the AIC value is, the better the model fits to the data [42]. For both compartmental models, the brain activity was corrected for the contribution of blood activity assuming a cerebral blood volume in the regions of interest fixed at 0.05 mL/cm 3 .
Furthermore, three RTMs were included in this study: the 2-step simplified reference tissue model (SRTM2) [43], the 2 parameter multilinear reference tissue model (MRTM2) [44] and the Logan reference tissue model [45]. For each of these models the possibility to reproduce the BP ND , obtained with the compartment models and the Logan plot, was analysed. Based on previous studies in humans [46] and rats [47], the cerebellum was used as a reference region. Furthermore, the BP ND was determined using a fixed k 2 ' value based on the mean k 2 ' of four high binding regions: hippocampus left, hippocampus right, presubgenual cingulate gyrus and subgenual cingulate gyrus. For the SRTM2 model and Logan reference model, regional coupling with the SRTM2 model was used to calculate the k 2 ' value. For the MRTM2 model, the MRTM model was used to calculated the k 2 ' value.

Test-retest analysis
To asses test-retest variability of the model parameters, one dog received two additional 60-minutes dynamic PET scans with bolus injection of 370 MBq [ 18 F]MPPF. BP ND were calculated using the three different RTMs (SRTM2, MRTM2 and Logan reference model) as described above.

Statistical analysis
Statistical analysis was computed using RStudio 1.1.456 using packages MASS (version 7.3-50) and Sommer (version 3.0). To compare the BP ND from the 2-TC models with those from the RTM, a multivariate linear mixed model with heterogeneous (unstructured) variances was set up on the data, containing BP ND as response variable and the delineated ROI's as outcome variable. The model included the individual animal as a random factor and the different kinetic models as fixed factor. The degrees of freedom were calculated based on the Welsh-Satterthwaite equation and the type-I error α was set at 0.001 (two-tailed) after Bonferroni correction (correction for the comparison between 15 regions and 4 different models). Furthermore, a random intercept was included. Finally, Pearson correlation coefficients (R 2 ) were calculated between all models using Microsoft Excel.

Radiosynthesis
The synthesis procedure of [ 18 ]MPPF gave rise to end of synthesis (EOS) activities of 1420 ± 650 MBq with high radiochemical and chemical purities of > 99%. The specific radioactivity measured with analytical HPLC was at least 101.3 GBq/μmol at EOS. [ 18 F]MPPF was rapidly metabolised to polar metabolites over the course of the PET scan. After 4 minutes, [ 18 F]MPPF accounted for 69 ± 10% of total activity in the blood plasma, 21 ± 4% after 10 minutes and declined to 7 ± 2% at 40 minutes. A Watabe function could be fitted to the fraction of parent tracer in plasma for each individual dog. Subsequently, a metabolite-corrected plasma input curve could be calculated in PMOD (Fig 1B).

Kinetic modelling
V T values (mean + SD) and corresponding BP ND in each ROI for the 1-TC and 2-TC model are shown in Table 1. The 1-TC and 2-TC model gave rise to maximum standard errors (SE) of 6.47% and 6.96%, respectively. The 2-TC model showed lower AIC-values in all of the observed brain regions compared to the 1-TC model. When plotting the BP ND of the 1-TC model and 2-TC model (Fig 4A, column 2), no general over-or underestimation could be found, which is illustrated with a mean difference around zero in the Bland and Altman plot. In addition, the BP ND obtained with the 1-TC and 2-TC model are well correlated with each other (R 2 = 0.979) (Fig 4A, column 1). Furthermore, the V T (SE MAX = 1.29%) and corresponding BP ND values obtained with the Logan plot are also shown in Table 1. BP ND -values obtained with the Logan plot were highly correlated with those from the 2-TC model (R 2 = 0.979) ( Fig  4B, column 1). Nevertheless, the Logan plot showed a moderate negative trend of differences, proportional to the magnitude of the measurement and mean underestimation of 7 ± 3% ( Fig  4B, column 2).
The BP ND of [ 18 F]MPPF estimated using the reference tissue methods (SRTM2, MRTM2 and Logan reference model) are shown in Table 2 and were plotted against those obtained with the 2-TC model (Fig 4C-4E). The BP ND values obtained with reference tissue models showed SE < 10%. No significant difference could be found between each of the different reference tissue models and the 2-TC model in any of the ROI's (p-values > 0.001), although, the Bland and Altman plots showed a mean underestimation of BP ND ranging from 7 ± 3% (MRTM2) to 11 ± 7% (SRTM2). However, all models are highly correlated with the 2-TC model (R 2 (SRTM2) = 0.982, R 2 (Logan Reference) = 0.999, R 2 (MRTM2) = 0.999) (Fig 4C-4E). Moreover, a small enlargement correlating to the BP ND levels for the Logan reference model and MRTM2 model could be seen in the Bland and Altman plots.

Test-retest variability
Test-retest variability for the regional BP ND using three different RTMs is represented in Table 3. The mean percentage difference in BP ND between test and retest ranges from 7.15%  (SRTM2) to 8.56% (Logan reference). The lowest difference is seen in the cortical regions and hippocampus, while highest difference is seen in the presubgenual cingulate gyrus, ACC and midbrain.

Discussion
To our knowledge, this is the first study that examines the use of [ 18 F]MPPF as PET-tracer to image the 5HT 1A receptor in the canine brain.
After a bolus injection of [ 18 F]MPPF, high radioactive uptake was found in all brain regions followed by a rapid washout (Fig 3). The pattern of [ 18 F]MPPF uptake into the brain corresponded well to the known distribution of the 5HT 1A receptor observed in human, rodent and cat studies [19,41,48]. The highest uptake of [ 18 F]MPPF was found in the hippocampus, anterior cingulate cortex, presubgenual cingulate gyrus and subgenual cingulate gyrus. As expected, low uptake was found in the cerebellum, indicating an absence of 5HT 1A receptors as previous reported and further approving its use as reference region in RTMs (Fig 2) [19,40]. Because the location of the raphe nuclei in dogs is not clearly defined in the literature and was very difficult discernible on the PET image, the nuclei were not included in this study. In accordance to the clinical study of Costes et al. [28], a rapid metabolism of [ 18 F]MPPF to polar metabolites was observed (Fig 1); 21 ± 4% of unmodified [ 18 F]MPPF was found after ten minutes and declined to 7 ± 2% at 40 minutes. Due to limited sensitivity of the analysing method, the metabolite correction curve needed to be extrapolated from 40 to 60 minutes using the fitted Watabe function.
In this study, the 1-TC, 2-TC and Logan plot blood input models were compared with three RTMs: SRTM2, MRTM2 and Logan reference tissue model. According to the lower AIC values of the 2-TC model compared to the AIC values of 1-TC in all ROIs, the 2-TC model showed a better fit to the data, especially in the regions with high radioactive uptake: presubgenual cingulate gyrus and subgenual cingulate gyrus, hippocampus and cingulate cortex. The Logan Table 1. Kinetic parameters derived from 1-TC model, 2-TC model and Logan plot. Distribution volumes (V T ), Akaike information criterion value (AIC) and binding potential (BP ND ) in all the ROIs derived from plasma input data using the 1-TC, 2-TC model and Logan plot. The data is expressed as mean ± SD. BP ND were estimated using the cerebellum as reference region, BP ND = (V T /V Ref ) -1. plot showed very similar V T values compared to the 2-TC model and the BP ND were highly correlated with each other (R 2 = 0.979), which indicates this graphical analysis could serve as a good alternative. Nevertheless, a moderate negative trend of differences and mean underestimation of 7 ± 3% should be kept in mind when using the Logan plot (Fig 4B, column 2). Calculating BP ND , using RTM, with the cerebellum as reference region, showed high correlation with the BP ND obtained by metabolite corrected plasma input 2-TC modelling (Fig 4). Although the Bland and Altman plots showed an underestimation of the BP ND values when using the RTMs compared to the 2-TC model, no significant difference was found in any of the ROI's. Furthermore, the MRTM2 model and reference Logan model show a dependency on the actual values. Nevertheless, the high correlation between the RTMs and 2-TC indicate the invasive blood sampling could be replaced by RTMs in future experiments with [ 18 F] MPPF in dogs, which requires no arterial catheterisation or blood sampling. Based on the highest correlation values (R 2 = 0.999) with the 2-TC model, the Logan reference model and MRTM2 model would be the models of choice, but a small enlargement correlating to the BP ND levels should be mentioned for both models. An excellent alternative would be the SRTM2 model, which doesn't have this dependency and showed also a very good correlation (R 2 = 0.982) with the 2-TC model.

Region
The test-retest variability was best for the SRTM2 model with a mean difference of 7.15%, compared to 8.27% and 8.56% for Logan reference model and the MRTM2 model, respectively. The cortical regions showed the lowest variability with a mean difference of 5.18%. High variability was seen in the presubgenuale gyrus and ACC for all three models. These regions are both very small which are highly susceptible to small errors in PET/MR fusion. High variability was also seen in the midbrain region but was due to very low BP ND -values.  TC model vs 2-TC model; 4B: 2-TC model vs Logan Plot; 4C: 2-TC model  vs Logan reference model; 4D: 2-TC model vs SRTM2 model; 4E: 2-TC model vs MRTM2. https://doi.org/10.1371/journal.pone.0218237.g004 Table 2. BP ND derived from the SRTM2-, Logan reference-and MRTM2 model. Binding potentials (BP ND ) in all the ROIs derived from RTM using the SRTM2-, Logan reference-and MRTM2 model. The data is expressed as mean ± SD.

SRTM2
Logan reference MRTM2 Potential minor effects of the anaesthesia on the kinetics of [ 18 F]MPPF during the PETscan should be mentioned, although the use of anaesthesia is inevitable in dogs and a widely used anaesthesia protocol was used. Currently very little is known about the potential effects of various anaesthesia protocols on the kinetic of PET-tracers in dogs and should be further investigated in the future. Also the use of only female dogs in this study should be addressed. A second limitation of this study would be the absence of an in vivo blocking study, demonstrating the selectivity and specific binding of [ 18 F]MPPF to the 5HT 1A receptor. However, this PET tracer has already proven its selectivity in various species and the high uptake brain regions are in accordance with other studies [19,41,48]. Furthermore, there is a 92% homology between the human 5HT 1A receptor and the canine 5HT 1A receptor [49]. Nevertheless, a future blocking study could provide a clear relationship between the model-derived parameters and the receptor expression levels.

Conclusion
This study describes the first step in the visualisation and quantification of the 5HT 1A receptor using a bolus injection of [ 18 F]MPPF in dogs. The results are consistent with the observations presented in the literature for other animal species and humans. The kinetics of [ 18 F]MPPF in the canine brain could be best described by a 2-TC model. Furthermore, for future experiments, compartmental modelling using invasive blood sampling could be replaced by RTMs, using the cerebellum as reference region. This could be of great value for future experiments analysing the function of the 5HT 1A receptor, improving both diagnosis and therapy in canine and human behavioural and neuropsychiatric disorders.