The intestinal permeability marker FITC-dextran 4kDa should be dosed according to lean body mass in obese mice

Animal studies

Three consecutive studies were conducted on a total of 45 DIO male C57BL/6 JCrlf mice (Charles River, France) and 70 lean male C57BL/6J mice (Janvier or Charles River, France) aged 8–16 weeks at arrival. Upon arrival, mice were fed either a HFD (D12592, Research Diets Inc.) or a LFD (D12450B, Research Diets Inc.) ad libitum. Mice were acclimatized in minimum 1 week before study initiation. DIO mice were single housed, 2 animals per cage with a partitioning wall between, while the control mice were housed 5 per cage in temperature (22 ± 2 °C) and humidity (50 ± 20 %) controlled rooms with a circadian rhythm (12 h light: 12 h dark; Lights on at 06:00). Mice had ad libitum access to food and water and enrichment in the form of chewing material, climbing racks, and nests provided in the cage. Bedding and enrichment materials were exchanged every week, and animals were inspected by caretakers daily. In the study period, humane endpoints were applied as follows; cases where the animals showed signs of permanent suffering, pain, or fear.

The care and use of mice in these studies were conducted according to national regulations in Denmark and with an experimental license granted from the Danish Ministry of Food, Fisheries, and Agriculture and conducted according to the EU Directive 2010/63/EU on the protection of animals used for scientific purposes.

Study design

For the studies investigating intestinal permeability in obese and lean mice, mice were weight stratified to the two subgroups, LBM dose or BW dose (lean groups, mean weight 27.2–27.3 g and obese groups mean weight 55,5–55,7 g, n = 10 /grp). Mice in the drug-response dose study weighed an average of 29,9 g and were weight stratified to receive a low or high dose of FD-4 (n = 10/grp). In the power corrected study (for power calculation, see Supplementary Table 1) using LM dose only, the average weight of DIO mice was 48,2 g (n = 25), while it was 28,7 for the lean mice (n = 23, two died before study termination). Mice scheduled to LBM dosing were scanned in an EchoMRI Body Composition Analyser (EchoMRI, Houston, TX, USA) the day before study termination. At study end, the mice were 18–24 weeks. On the study day, mice were weighed and fasted from 6 AM to 10 AM before FD-4 (dissolved in PBS at a concentration of 125 mg/ml) PO dosing using a stomach tube (for study design, see Supplementary Fig. 1). After retroorbital blood sampling, mice were sacrificed, and the liver and gastrointestinal tract were sampled. All experiments were repeated two to three times and carried out by trained and licensed personnel. Experimenters were not blinded, only at analysis.

In vivo intestinal permeability test

Mice fasted for four hours before FD-4 (Sigma-Aldrich, USA) were administered orally (600 mg/kg) in a randomized order. Two hours after dosing, mice were anesthetized using isoflurane (induction 5%, maintenance 2% isoflurane, 0.7 L/min N2O, 0.3 L/min O2), and retro-orbital blood sampled in K3-EDTA-coated tubes (Sarsted, Germany) followed by cervical dislocation. The samples were centrifuged (4 °C, 7 min, 8000 g), and plasma was collected in clear Eppendorf tubes (Eppendorf AG, Hamburg). Plasma from PBS dosed mice were used for the standard curve. FD-4 concentrations in plasma were analyzed in duplicates using a spectrophotometer (SpectraMax M4, Molecular Devices, San Jose, CA, USA) with excitation λ 485 nm and emission λ 535 nm.

qPCR

The jejunum was sampled immediately after termination, snap frozen, and stored at −80 °C for qPCR analysis. All samples were homogenized in 1 ml Trizol (#15596018 Invitrogen), and RNA was extracted using Rneasy mini kit (#74106, Qiagen) with Dnase treatment included (79254, Qiagen) and analyzed by NanoDrop 8000 system (ThermoFisher Scentific). The total amount of RNA isolated was approximately 10 µg per jejunum. Iscript (#1708841 Bio-Rad) was used to prepare cDNA, and the qPCR analysis was performed with TaqmanTM Fast Advanced Master Mix (4444964, Applied Biosystems) and the listed Taqman gene expression assays (see Supplementary Table 2). The genes of interest were normalized to Sdha and presented as fold-change of gene expression (2–ΔCt) compared to the lean mouse group. At analysis, the samples were blinded to the analyst.

Statistical analysis

GraphPad Prism version 9.0.1 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis; p values below 0.05 were considered significant. Group comparisons were made using a t test or One-way ANOVA. If the data did not show Gaussian distribution by D’Agostino-Pearson test or variances were unequal by the Brown-Forsythe test, the data were log-transformed. Pearson’s correlation analysis was used to test the statistical relationship. Results are shown as mean ± standard deviation.

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