Semaglutide Attenuates Neuroinflammation in Mice
Semaglutide Attenuates Neuroinflammation in Mice
Immediately after termination, terminal plasma was collected, and the whole brain was dissected and divided into left and right hemisphere and placed in liquid nitrogen and stored at -80°C. Hippocampus: Frozen tissues from the hippocampi were mechanically dissociated in a 2mL glass tissue douncer (Sigma, cat. no.: D8938 with pestels) using 1mL Nuclei EZ Lysis Buffer (Sigma, cat. no.: NUC101-1KT) and 10 firm strokes with glass pestle B. Subsequently, the homogenized samples were incubated on ice for 5 min and transferredthrough a 40µM cell strainer (PluriSelect, cat. no.: 43-10040-40) to a 2mL Protein LoBind tube (Sigma, cat. no.: EP0030108132). The samples were centrifuged (Eppendorf 5810R) for 10 min at 1000g with brake set to 1 and resuspended in 250µL Nuclei Buffer (1% BSA (Sigma, cat. no.: SRE0036), 2mM MgCl2 (Sigma, cat. no.: M1028), 0.04U/µL Protector RNase inhibitor (Sigma, cat. no.: 3335399001) in PBS, pH 7.4 w/o Ca2+, Mg2+ (Gibco, cat. no.: D1556)). The samples were further subjected to an Iodixanol gradient purification, where 250µL of 50% Iodixanol/nuclease-free water (Sigma, cat. no.: D1556) was added, and the samples were thoroughly, but gently, mixed by pipetting and layered on top of 500µL 29% Iodixanol followed by centrifugation for 22min at 14,000g with brake set to 1. The isolated nuclei were located at the bottom of the tube and resuspended in 500µL Nuclei Buffer and incubated on ice for 15 min.  Samples were centrifuged for 10 min at 1000g with brake set to 1 and resuspended in 100µL Nuclei Buffer with 0.5µg TotalSeqTM-A anti-Nuclear Hashtag (HTO) for multiplexing and 0.5µg NeuN Alexa Flour 488 (Millipore, cat. No.: MAB377X). Samples were then transferred to a 1.5mL Protein LoBind tube (Sigma, cat. No.: EP0030108116) and incubated on ice for 30 min. Samples were again centrifuged for 10 min at 1000g with brake set to 1 after which samples were resuspended in 200µL Nuclei Buffer with 0.4µg 7-AAD (Sigma, cat. No.: 3462) for sorting.  DVC: Frozen tissues from DVCs were isolated as described for the hippocampal samples and resuspended in 500µL Nuclei Buffer without subsequent Iodixanol gradient purification.   Samples were then centrifugated as described for the hippocampal samples and resuspended in 100µL Nuclei Buffer with 0.5µg TotalSeqTM-A anti-Nuclear Hashtag antibody for multiplexing73, transferred to a 1.5mL Protein LoBind tube (Sigma, cat. no.: EP0030108116) and incubated on ice for 30 min. Following centrifugation (as above), the sample was resuspended in 200µL Nuclei Buffer with 0.4µg 7-AAD (Sigma, cat. no.: 3462) for sorting.   A total of 80 6-8-weeks old male C57BL/6J mice (Charles River, Germany) were singled housed (2 mice/cage separated with divider) upon arrival, and 8-10 weeks old at study start (n =7-8/group). Mice were dosed with semaglutide (30 nmol/kg, SC) or vehicle (pH7.4; 0.007 % polysorbate 20; 50 mM phosphate; 70 mM sodium chloride). Mice dosed with semaglutide were uptitrated to the full dose by giving them 3 nmol/kg on the first day of dosing, 10 nmol/kg on the second day of dosing, followed by the full dose of 30nmol/kg daily for the remainder of the study, which is a clinically meaningful dose and within a dynamic dose response 40. Mice were dosed with semaglutide or vehicle in the morning between 7-9 am. Two weeks after the onset of dosing with either semaglutide or vehicle, mice were dosed with LPS or PBS for a total of 3 days. On the days of LPS/PBS dosing, animals received their dose of semaglutide or vehicle one hour before the administration of LPS/PBS. The dose of LPS was 1.0 mg/kg daily IP. Mice were euthanized either 2 hours, 24 hours, 5 days or 11 days after the last dose of LPS/PBS Hippocampus: From each sample we aimed to 4,000 7-AAD-positive nuclei (SONY SH800S cell sorter) using a 70µm sorting chip (Sony Biotechnologies, cat. No.: LE-C3207) into a 2mL Protein LoBind tube with 18.8µL RT Reagent B (10X Genomics, Chromium Next GEM Single Cell 3' Kit v3.1, cat. No.: PN-1000268). Of the 4,000 sorted nuclei, 2,000 were NeuNlow. DVC: From each sample, 2,500 7-AAD-positive nuclei were sorted (SONY SH800S cell sorter) with a 70µm sorting chip (Sony Biotechnologies, cat. no.: LE-C3207) into a 2mL Protein LoBind tube with 18.8µL RT Reagent B (10X Genomics, Chromium Next GEM Single Cell 3' Kit v3.1, cat. no.: PN-1000268). Following sorting, the volume was adjusted to 43.1µL with Nuclei Buffer and the final GEM Master Mix reagents were added as per manufacturer's procedure, which was followed from then on for library preparation with dual indexing.    HTO libraries were prepared by following the procedure from BioLegend and quantified by Qubit and TapeStation (Agilent TapeStation 4200 System) with TapeStation High Sensitivity D1000 DNA (Agilent, cat. no.: 5067-5584 and 5067-5585).