Animals

All animal experiments complied with the European Communities Council Directive (2010/63EU) and the German Animal Protection Law and were approved by the Tübingen Animal Protection Committee (Regierungspräsidium Tübingen, Germany). The rats were housed in groups of 4 with a 12 h light/dark cycle at a temperature of 22 °C and relative humidity between 40 and 60%. Food and water were obtainable ad libitum. This research used 20 female and 4 male Sprague Dawley rats (286.5 ± 36.8 g) purchased from Charles River Laboratories (Sulzfeld, Germany). For an overview of the different experimental groups please see Table 1.

Table 1 Experimental groups and [11C]raclopride injected and molar radioactivities at the time of injectionExperimental Design

[11C]raclopride PET measurements were performed under three different respiratory conditions and anesthesia protocols to determine the influence on [11C]raclopride kinetics.

To investigate the impact of mechanical ventilation on the [11C]raclopride kinetics, all the rats were anesthetized with 0.5% isoflurane and medetomidine (absolute bolus 0.08 mg/kg and 0.15 mg/kg/h ip infusion). The rats were divided into the following groups:

(1)

no ventilation nor pancuronium bromide infusion (MED-V-P).

(2)

with ventilation and no pancuronium bromide infusion (MED+V-P).

(3)

with ventilation and pancuronium bromide infusion (MED+V+P).

To determine the effect of anesthetics on the [11C]raclopride kinetics, two additional anesthesia protocols were investigated. Both of these were paired with mechanical ventilation and pancuronium infusion:

(4)

1.3% isoflurane (ISO+V+P).

(5)

α-Chloralose initiated with an absolute bolus of 42 mg/kg, followed by an infusion rate of 20 mg/kg/h ip infusion (AC+V+P).

Group 3, 4 and 5 received a constant ip infusion of pancuronium bromide of 1 mg/kg/h. Animals were ventilated with a breathing machine with 60 breaths/min of respiratory rate, 60% of inspiration and 0.5 ml/min of air in groups 2, 3, 4, and 5.

Radiotracer Synthesis

[11C]Raclopride was synthesized as previously described [24, 25] (details are provided in the supplemental information).

Animal Preparation and [11C]raclopride PET Imaging

PET imaging was performed on a dedicated small animal Inveon PET scanner (Siemens Healthcare, Knoxville (TN), USA). The rats were placed in a knock-out box, and 3% isoflurane evaporated in regular air was delivered for anesthesia induction through the small animal veterinary anesthesia machine (Landmark Anesthesia System, Vetland, Louisville (KY), USA). Once anesthetized, each rat weight was recorded, and the isoflurane concentration was reduced to 2% for subsequent procedures.

A 30 G needle catheter was inserted into a tail vein for the [11C]raclopride bolus injection. A second catheter was placed intraperitoneally for the constant infusion of the corresponding anesthetics (MED-V-P, MED+V-P, MED+V+P and AC+V+P). All groups except MED-V-P were endotracheally intubated using a 14 G plastic tube and connected to a small animal ventilator (DC1 73–3629, Harvard Apparatus, Holliston, MA, USA) connected to isoflurane and air. The respiration rate was set to a constant rate of 60 breaths per minute.

Rats were then positioned in the center of the PET scanners field of view on carbon beds with a water heated mat. The temperature of the rats was constantly monitored using a rectal probe and maintained at about 37 °C by a temperature feedback-controlled system (Medres, Cologne, Germany). At least thirty minutes prior to the PET acquisition, the isoflurane concentration was reduced to 1.3% (ISO+V+P), 0.5% (MED-V-P, MED+V-P, MED+V+P), and 0% (AC+V+P) in regular air and kept constant during the whole time-course of the experiment.

[11C]Raclopride was injected intravenously as a fast bolus using a computer-controlled infusion pump (Harvard Apparatus PHD 2000 Infusion Pump) at a speed of 1000 µl/min for 30 s with an activity concentration of 100 MBq/ml. Dynamic PET data acquisition was started 5 s before tracer injection and was continued for 60 min followed by a 13-min transmission measurement with a cobalt- 57 point source for attenuation correction. Dynamic PET data was divided into 39 time-frames (12 × 5 s, 6 × 10 s, 6 × 30 s, 5 × 60 s, 10 × 300 s). Images were reconstructed using a 3D ordered subset expectation maximization (OSEM) algorithm using a matrix size of 128 × 128 × 63 resulting in a voxel size of 0.388 × 0.388 × 0.796 mm3.

Image Analysis

PET image analysis was carried out using PMOD 4.2 software (PMOD Technologies, Zürich, Switzerland). To obtain quantitative images, dead time, decay correction and normalization were applied. To avoid motion during the PET imaging session, the head was fixed with removable tape. PET images were aligned to a reference MR rat brain atlas with volumes of interest (VOI) matching the atlas [26]. Time activity curves (TACs) of both the left and right caudate putamen (CPu) and the cerebellum were derived and converted into standardized uptake values (SUVs), calculated using the following equation:

$$SUV \left(t\right)= \frac)})}$$

The SUV ratio was then calculated as SUVR- 1 from individual TACs using the cerebellum (CER) as a receptor-free reference region in the following equation:

The peak [11C]raclopride activity, as well as the time taken to reach this peak in the CPu and cerebellum, were obtained from the TACs. The delivery rate of the tracer was determined by the quotient of the peak [11C]raclopride activity and the time to peak values.

Statistical Analysis

Data followed a normal distribution, comparisons were performed by one-way ANOVA with false discovery rate (FDR) correction (see supplementary methods for details).