[¹⁸F]MPPF and [¹⁸F]FDG μPET imaging in rats: impact of transport and restraint stress

Thirty-two female Sprague Dawley rats (244.0 ± 10.8 g, Envigo, the Netherlands) were used for this study. All animals were housed individually under controlled environmental conditions (22-24 °C, 45-60% humidity, TECNIPLAST GR 1800 Double Decker) in a 12-h dark-light cycle with ad libitum access to food (Ssniff Spezialdiäten GmbH, Soest, Germany) and tap water. Animals were allowed to acclimatize to the housing facility for 7 days. During this acclimatization phase, animals were habituated to handling by the experimenter with handling sessions every second day for 2-3 min. Once a week, all animals received a clean cage with new bedding material (Lignocel® Select, J. RETTENMAIER & SÖHNE GmbH + Co KG, Rosenberg, Germany) and 14 g of new nesting material (Enviro Dri®, Claus GmbH, Limburgerhof, Germany). During the entire study, the health condition of the animals was checked daily and body weight was measured every third day. The study was approved by the government of Upper Bavaria (license number: AZ 55.2-2532-17-86) and was planned and conducted in line with the German Animal Welfare Act and the EU directive 2010/63/EU. All procedures and reporting were performed according to the ARRIVE guidelines and the Basel Declaration including the 3R concept.

Rats were randomly allocated to one of three groups (www.​randomizer.​org): transport + restraint (11), transport (11), and naive (10). For twelve consecutive days, animals in the transport + restraint and the transport groups were removed from their home cage and transported (in a separate transport cage) from the animal facility to a laboratory (distance of < 5 meters). Here, rats of the transport + restraint group were placed in a plexiglass tube with ventilation holes (16 cm in length and 9 cm in diameter) for 1 h (from 10:00 am to 11:00 am) and transport animals were housed individually in a cage with bedding and nesting material. The experimenter was not present in the procedure room during the 1-h exposure phase. Following 1 h, all animals of the transport + restraint and the transport group returned to the animal facility. Naive animals remained in their home cage in the animal facility and were only handled for cage cleaning or weighing during this period. The timeline for the assessment of nest building, saccharin preference test, [¹⁸F]FDG and [¹⁸F]MPPF PET imaging, serum corticosterone, and adrenal gland weight of all animals is illustrated in Fig. 1.

On day 13, all animals underwent PET imaging with [¹⁸F]FDG. Small animal PET imaging was performed on a Siemens Inveon DPET scanner (Siemens Medical Solutions, Munich, Germany). Anesthesia was induced by inhalation of isoflurane (4% for induction and 2% for maintenance during preparation, injection, and scanning) supplemented with oxygen at a rate of 1 l/min. [¹⁸F]FDG was administered via a tail vein cannulation and two rats were simultaneously scanned head-to-head. Rats were fasted overnight to reduce endogenous glucose levels and an emission scan of 15 min was acquired 30 min post tracer injection. The average injected radioactivity was 32.2 ± 7.1 MBq in a volume of 0.5 mL. On day 15, a subset of animals underwent PET imaging with [¹⁸F]MPPF. Twenty animals were scanned (naive = 7, transport = 6, transport + restraint = 7) with three productions of [¹⁸F]MPPF. One production of [¹⁸F]MPPF had an inadequate specific activity and data from these five animals scanned with this batch could not be considered in the analysis resulting in n = 5 for all treatment groups. The average injected radioactivity of [¹⁸F]MPPF was 25.9 ± 6.3 MBq in a volume of 0.5 mL and an emission scan of 50 min was acquired 10 min post tracer injection. The molar activities were in the range of 37-111 GBq/μmol, and the radiochemical purities, as determined by radio-HPLC, were > 96%. All emission scans were either preceded or followed by a 15 min transmission scan using a rotating Cobalt-57 [⁵⁷Co] point source. Data were acquired in list-mode and reconstructed into one frame for [¹⁸F]FDG and eleven frames (5 × 60 s, 3 × 300 s, 3 × 600 s) for [¹⁸F]MPPF. All PET data were corrected for attenuation, scatter, and decay. Reconstruction was performed with four ordered-subset-expectation-maximization 3D iterations and 32 maximum-a-posteriori 3D iterations and a zoom factor of one resulting in a final voxel dimension of 0.783 × 0.783 × 0.8 mm.

Image processing was completed using the PMOD v3.4 software (PMOD Technologies Ltd., Zurich, Switzerland). Tracer-specific PET templates were generated through the averaging of PET images previously aligned by manual co-registration to a digital cryosection-based atlas of the rat brain [22, 23]. Co-registered PET images were then spatially normalized into the space of their respective tracer-specific template by the PMOD brain normalization tool (equal modality; smoothing by 0.8 mm, non-linear warping; 16 iterations, frequency cut-off 3, regularization 1.0, no thresholding) [24]. Volumes of interest (VOIs) were defined as previously described [20]. VOIs for the parietal cortex (16 mm³), medial prefrontal cortex (13 mm³), thalamus (22 mm³), amygdala (23 mm³), striatum (15 mm³), hypothalamus (11 mm³), cerebellum (82 mm³), and pons (8 mm³) were drawn manually with reference to the cryosection atlas (Supplementary Fig. 1). For visualization purposes, images of mean tracer uptake per treatment group were generated, smoothed with an isotropic Gaussian filter (0.5 mm), and projected onto the cryosection atlas of the rat brain.

[¹⁸F]FDG uptake was quantified as the standardized uptake value ratio (SUVR) by normalizing tracer uptake in target regions to that of the pons.

For [¹⁸F]MPPF, the non-displaceable binding potential (BP_nd) was calculated in target regions from the entire scan [25] using the Logan linear graphical method [26] with the cerebellum as a reference tissue input. Threshold-based automatic VOIs were generated from [¹⁸F]MPPF uptake for the hippocampus (61 mm³) and septum (9 mm³) due to the high specific binding in these regions.

Nest building constitutes a non-essential behavior in an animal-facility environment, which can be compromised by distress.

Following the first session in the laboratory, all animals were placed in a clean cage with the addition of new nest material (14 g Enviro Dri®, Claus GmbH, Limburgerhof, Germany). Starting on day 2, the complexity and shape of nests (1 = flat, 2 = slightly curved, 3 = deep) was scored daily between 8 and 9 am for 7 days, at which point all cages were cleaned with the addition of fresh nesting material. Nest scoring was then continued until day 13. The scoring system is illustrated in Supplementary Fig. 2.

The saccharin preference test was used to assess anhedonia-like behavior. A two-bottle testing paradigm was performed over 4 days. On the first and third day, both bottles were filled with tap water. On the second and fourth day, one bottle contained a 0.1% saccharin solution (Aldrich Saccharin ≥ 98%, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). To exclude a possible side preference, the placement of the saccharin-containing bottle was alternated from the right (second day) to the left (fourth day) side of the cage. Saccharin preference was analyzed according to a protocol published by Klein et al. [27]. Each bottle contained 300 g of liquid and the consumption over 24 h was measured. The saccharin preference was calculated as saccharin intake/total fluid intake (water + saccharin) × 100. The saccharin test was performed twice to assess possible differences between the early (day 3-6) and late (day 9-12) phase of the restraint stress procedure.

On day 16, all rats were killed by an overdose of pentobarbital (600 mg/kg i.p., Narcoren, Merial GmbH, Hallbergmoos, Germany). Blood samples were obtained by cardiac puncture. Adrenal glands were removed and weighed.

Statistical analysis was performed with GraphPad Prism (Version 5.04; GraphPad, San Diego, CA, USA) and R version 3.4.3 [28]. Differences between groups were assessed by one-way analysis of variance (ANOVA) followed by a Bonferroni multiple comparison post hoc test or by repeated-measures two-way ANOVA with Tukey HSD post hoc comparisons. Normal distribution was confirmed by a Shapiro-Wilk test. Ordinal data (nest-building scores) were analyzed using the Kruskal-Wallis test followed by Dunn’s multiple comparison post hoc test. Spearman correlation matrix and graphs were calculated and visualized using the R package “corrplot” [29]. Data sets from animals with missing data were not considered for individual correlation analyses. Nest building scores are presented as the median score per day. All other data are shown as mean and standard error of the mean (SEM).

5-HT_1A receptor availability was assessed by [¹⁸F]MPPF μPET on day 15, i.e., 3 days following the last exposure to transport or to transport and restraint (Fig. 2a and b). The regions of interest were selected based on information about regional 5-HT_1A receptor expression and the average parametric maps of [¹⁸F]MPPF binding potential (Fig. 2a).

A relevant [¹⁸F]MPPF binding was detected in all regions of interest comprising the medial prefrontal cortex, septum, and hippocampus (Fig. 2b). In all groups, the highest level of binding was observed in the hippocampus.

In the septum and medial prefrontal cortex, [¹⁸F]MPPF binding did not differ between groups (F (2, 12) = 1.256; F (2, 12) = 0.024; Fig. 2b). In rats with transport to the laboratory, hippocampal [¹⁸F]MPPF binding exceeded that in the transport and restraint group and in the naive group (F (2, 12) = 4.759, p = 0.030; post hoc p = 0.016; Fig. 2b).

Glucose metabolism was studied based on [¹⁸F]FDG μPET at day 13, i.e., 1 day following the last exposure to transport or to transport and restraint. Averaged [¹⁸F]FDG metabolic maps for all groups are shown in Fig. 2c.

Comparison between groups did not reveal any relevant group differences in all regions of interest (for all regions: F(2, 27) = 0.233-1.790, Fig. 2d).

[¹⁸F]FDG uptake was quantified with the pons as a reference tissue. We confirmed that [¹⁸F]FDG uptake in this region did not significantly differ between treatment groups (Supplementary Fig. 3). Two naive rats were excluded from [¹⁸F]FDG analysis due to a paravenous injection.

Body weight was determined on days 1 (= baseline), 3, 6, 9, and 12 of the study immediately prior to transport. The percent weight change at all time points was calculated in relation to baseline level (Fig. 3a).

Variance analysis revealed a significant interaction between the effect of “treatment” and “time” on body weight changes (F (6, 87) = 3.045, p = 0.0095). A difference to naive control animals became evident at day 9 for the group with transport to the laboratory, and at day 9 and 12 for the group with transport and restraint (post hoc p = 0.033; p = 0.038; p = 0.043).

The shape and complexity of nests was scored daily from day 2 until day 13. The comparison of scores between groups did not indicate relevant group differences (Supplementary Fig. 4).

The saccharin preference test was performed twice during the study. All groups exhibited a pronounced preference for the saccharin solution at both time points investigated (Fig. 3b). The ratio between water and saccharin consumption was in the same range in all groups, during both the early and the late test phase (early phase: F (2, 28) = 0.317; late phase: F (2, 28) = 0.568).

Serum corticosterone levels and adrenal gland weight were determined at the end of the study to assess a possible activation of the hypothalamic-pituitary-adrenal (HPA) gland axis.

Both, corticosterone levels and adrenal gland weight were in the same range in all groups (F (2, 29) = 0.6777; F (2, 29) = 0.8931; Supplementary Fig. 5A and B).

In order to obtain information about the link between μPET data and clinical, behavioral, or biochemical data, we completed a cross-correlation analysis (Supplementary Fig. 6).

A moderate correlation was shown between body weight change at day 9 and [¹⁸F]MPPF binding in the hippocampus and medial prefrontal cortex (r = −0.64, p = 0.01; r = −0.50, p = 0.057; Fig. 4).