Transcriptomics

RNA transcriptomics was performed on isolated human islets and exocrine tissue from five donor pancreas stored at – 70 ° C, as well as fresh frozen pancreatic tissue included in an OCT compound (Sakura Finetek, Alphen aan den Rijn , Netherlands). The use of human tissue from Uppsala Biobank (record # 827) has been approved by the Regional Ethics Board, Uppsala, Sweden (now Swedish Ethics Review Authority) (2011/473, Ups 02-577, 2015 / 401) and have been anonymized, collected, and processed in accordance with Swedish local and national institutional rules and regulations. The Uppsala Regional Ethics Council waived the need for informed consent.

RNA was extracted using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Breaking was performed using a 3mm steel grinding ball (VWR, Radnor, PA) and a vortex. RNA concentration and integrity (RIN) were determined by the Qubit 2.0 Fluorometer (Life Technologies) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively. The purity of the samples was confirmed by an A260 / A280 value greater than 2.0 using a Nanodrop (Thermo Scientific, Wilmington, DE). Samples with RIN values ​​greater than 7.5 were sequenced by Illumina HiSeq2000 and 2500 (Illumina, San Diego, CA) using the standard Illumina RNA-seq protocol with a read length of 2 × 100 bases. Details of the analysis of mRNA levels in this dataset have been described previously. ¹⁹.

Radiosynthesis of [¹¹C]UCB-J

[¹¹C]UCB-J was synthesized using a commercially available precursor compound (Pharmasynth AS) and a one-step synthesis method in THF / water and formulated in 10% ethanol in a phosphate saline (PBS) as indicated above²⁰. With this method, the radioactivity yield of [¹¹C]UCB-J was 1500 ± 600 MBq, molar activity 340 ± 190 MBq / nmol and radiochemical purity> 99.9% at the end of synthesis (n = 15).

In Vitro Autoradiography Binding Studies

Adjacent frozen sects. (10 µm) of rat and pig pancreas as well as of rat brain and excised INS1 xenografts (insulinoma / beta cell model) were prepared on a cryostat (Cryostar NX70, Thermo fisher scientist) at – 20 ° C and mounted on adhesive glass slides (Superfrost Plus, Menzel-Gläser, Germany) to be stored at – 20 ° C. For in vitro autoradiography (ARG), frozen slides were first preincubated in phosphate buffered saline (150ml PBS, pH 7.4) at room temperature for 10 min and washed with MQ water for 1 min. Following, [¹¹C]UCB-J in PBS and ethanol (1 MBq / ml) was added and sections were incubated for 40 min. To remove excess and unbound buffer [¹¹C]UCB-J, the slides were washed three times in PBS and once in MQ water and dried in a laboratory oven (Termaks, Bergen, Norway) for 10 min. A set of calibration standards was created for quantification by pipetting 10 µl of drops from the same stock onto absorbent chromatography paper. The slides were then exposed to a freshly erased storage phosphor screen (BAS-MS, Fujifilm) for two half-lives of. ¹¹C (40 min) then scanned on a phosphorus imager (Amersham Typhoon FLA 9500 Phospor Imager, GE) with a sensitivity of 4000 and a pixel size of 25 µm. Images were analyzed in ImageJ software (National Institutes of Health, US).

Immunolabelling for insulin

Adjacent sections of frozen in vitro autoradiography were immunostained for insulin expression. Sections were fixed in acetone (4 ° C) for 5 min, allowed to dry for 15 min, then washed in PBS and Dako wash buffer. Donkey serum (3% in PBS) was added for blocking and incubated for 20 min at room temperature, followed by overnight incubation with 1: 400 guinea pig anti-insulin (Fitzgerald , Acton, MA, USA). ref. 20-IP30) at 4 ° C. The next day, sections were first washed in 2 × 5 min PBS and 1 × Dako wash buffer. Donkey anti-guinea pig secondary antibody conjugated to Alexa 488 (1: 300, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Code No. 706-545-148) were added to the sections for 50 min at room temperature. Then the sections were rinsed 3 × 5 min PBS then incubated with DAPI for 10 min to obtain a background. Finally, sections were washed 2 × PBS and mounted in fluorescence mounting medium on protective glass.

Animal handling

The procedures involving animal testing have been approved by the Animal Ethics Committee of the Swedish Animal Care Agency and performed in accordance with ARRIVE and institutional guidelines (“Uppsala University Guidelines for Animal Testing” , UFV 2007/724).

PET-MRI imaging of rats

The distribution of the tracers was examined in Sprague – Dawley rats (SPRD). The rats were anesthetized as described above and placed on a heating pad under the camera immediately after the [¹¹C]UCB-J injections (22 MBq to 74 MBq). A 60 min dynamic nanoPET-MRI analysis (Mediso Medical Imaging Systems, Hungary) on the pancreas (spatial resolution 1.5 to 2.0 mm²¹, 212 × 212 × 239) was acquired with short axial T1-weighted (TR / TE 497 / 9.45 ms) and coronal (TR / TE 481 / 9.45 ms) sequences (0.4 mm spatial resolution) , 63 slices) before a 30 min scan on the brain (TR / TE 300 / 9.45 ms, 38 slices), either at inclusion (n = 4) or after preblocking (n = 3) with 30 mg / kg LEV in 0.9% NaCl (10 mg / ml) 30 min before the start of the scan. The rats were euthanized with CO₂ after the scans. PET images were reconstructed for attenuation and dispersion correction using Tera-Tomo â„¢ 3D (OSEM, Monte Carlo DOI estimation, correction for attenuation, dispersion, random values ​​and time dead to uniform positron range: 4 iterations, 6 subsets, voxel size 0.4 mm³, 0.3 mm³ resolution) reconstruction.

Ex vivo autoradiography

An ex vivo autoradiography was performed on rats (n = 8) injected with [¹¹C]UCB-J. Briefly, the rats were anesthetized with isoflurane (5% initially and then 3% to maintain anesthesia), and [¹¹C]UCB-J was administered as a single injection (46 MBq to 184 MBq, corresponding to a similar mass of tracer) via the tail vein. The rats were sacrificed by CO₂ 20 (n = 5), 40 (n = 1), 60 (n = 1) or 80 (n = 1) min after injection. Two of the rats euthanized 20 min after injection were pretreated with the LEV blocking agent (30 mg / kg) before the radioactive compound.

Immediately after euthanasia, the brain, pancreas, and spleen were excised, incorporated into OCT medium, and frozen. Coronal sections of pancreas and spleen and axial sections of 20 µm thick brain sections were cut using a cryostat (Cryostar NX70, Thermo Fisher Scientific). Sections were mounted on Superfrost Plus microscope slides (Menzel-Gläser, Germany) and exposed in the same manner as described above against a phosphorus imaging plate (BAS-MS) for 40 minutes at room temperature.

Pig PET-CT Imaging

Healthy male and female pigs reared locally (n = 3.26 ± 3 kg) were placed supine on a table and anesthetized with fentanyl 5-10 mg / kg IV and maintained with a continuous IV infusion of ketamine 30 mg. / kg / h, midazolam 0.1-0.4 mg / kg / h and fentanyl 4 mg / kg / h. Pig’s vital signs (blood sugar, oxygen saturation, ECG, blood pressure, body temperature, and end-of-expiration carbon dioxide (ETCO)₂)) were monitored throughout the study and fluid homeostasis was maintained with continuous infusion of Ringer’s acetate (10 ml / kg / h) and 0.9% NaCl (Fresenius Kabi AB , Sweden). An arterial catheter was inserted into the right carotid artery for blood collection and blood pressure measurement, and an introducer was placed over a central venous catheter for administration of. [¹¹C]UCB-J and contrast media.

First, an attenuation CT scan (100 kV, 80-400 mA, noise index 10, rotation 0.5 ”, full spiral, section thickness 3.75 mm, pitch 0.98: 1, diameter recognition distance 50 mm) was acquired using a 4-ring digital ring. system, computed tomography 64 slices with an axial field of view (FOV) of 198 mm. A 90 min dynamic PET scan (4 mm spatial resolution, 33 images: 12 × 10 ”, 6 × 30 ”, 5 × 2 ‘, 5 × 5’, 5 × 10 ‘) (Discovery MI, GE Healthcare) on pancreas and a 30-minute static brain scan were performed after bolus injection of [¹¹C]UCB-J (260-350 MBq). The scans were repeated after IV pretreatment (1 h before injection of the tracer) of LEV 20 mg / kg in 100 ml of 0.9% NaCl by slow infusion (400 ml / h), and a new bolus of [¹¹C]UCB-J was administered approximately 4 hours after the first injection. Arterial blood samples were acquired during dynamic analyzes to obtain the volume of the tracer in both plasma and whole blood by measuring activity on a gamma-well counter. The pancreas was visualized independently by late arterial (15 s) and venous CT with phase contrast (Omnipaque 350 + 40 ml NaCl, 3.5 ml / s, followed by the bolus at the threshold of the descending aorta 100 HU) to facilitate segmentation of PET images. PET images were reconstructed using an iterative VPFX-S (GE Healthcare) algorithm (OSEM, Time of Flight, Resolution recovery: 3 iterations, 16 subsets, 3 mm postfilter and 256 × 256 Matrix).

PET data analysis

The volumes of interest were manually defined on the organs on corrected transaxial projections SUV using the PMOD software (PMOD Technologies LLC, Zurich, Switzerland). Regional temporal activity curves (TACs) were obtained by segmenting the pancreas, brain, and adrenal glands for target organs and spleen and muscles as negative references. Graphics were presented on Microsoft Excel (Microsoft Office) and GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). All data is presented as the mean and standard deviation of the mean. Comparisons between the initial and blocking studies were assessed by Student’s t-test (two-tailed), where p