Experiment 1
Animals
Each animal received saline by i.p. injection (0.9% (w,v) NaCl) or liraglutide (2.5 nmol/kg body weight (bw), 25 nmol/kg bw or 250 nmol/kg bw) (female, C57/BL 6 background, n = 5 per group). At 5 min, 30 min or 3 h post treatment the mice were painlessly killed with an overdose of anaesthetic. Subsequently, animals were perfused with PBS in order to clear the circulation of all blood. Each brain was weighed, placed in a Bijou tube, snap frozen in liquid nitrogen and stored at -80 C until further processing and analysis. All experiments were licenced by the UK home office (Project licence No. PPL2603b).
Acid ethanol extraction of liraglutide from mouse brains
Tubes containing the brains from the mice were removed from storage and kept in liquid nitrogen. Each tube was processed one at a time to avoid thawing. 2.5 ml of acid ethanol (75% ethanol, 1.5% conc HCl)/g of tissue was added and the sample sonicated using a Soniprep 150 plus HSE (Davidson and Harley Ltd). Samples were stored on ice before centrifugation at 10,000 rpm for 15 min at 4 C using the Beckman centrifuge. The supernatant was poured off into fresh eppendorf tubes. 300 μl of the supernatant was removed to a clean eppendorf tube and 300 μl of 1 M Tris (pH 7.6) was added. These samples were then dried down in a Speedvac at 45 C for 1 h 30 min to completely remove the liquid. Samples were reconstituted in GLP-1 ELISA kit assay buffer (Millipore, MA, USA).
ELISA analysis of brain samples
The GLP-1 (Active) 96 well fluorescent ELISA kit (EGLP-35 K, Millipore, MA, USA) was used to measure the Liraglutide levels detected in the brain samples. The assay was performed according to the manufacturers instructions. Briefly, the plate was incubated with wash buffer for 5 min then decanted and the excess removed by tapping on absorbent towels. Assay buffer was added to all wells followed by either the standards, quality controls or samples in the appropriate order on the plate. The plate was covered with a plate sealer, gently shaken then incubated overnight at 4°C. On day 2 the plate was washed 5 times with wash buffer, detection conjugate added and incubated at room temperature for 2 h. The plate was then washed 3 times and diluted substrate added and allowed to incubate in the dark for 18 min. Stop solution was added and the plate read at 355 nm/460 nm using the Flexstation 3 and Softmax programme. Brain samples were diluted 1:10 prior to measurement on the plate so results were adjusted accordingly following analysis.
cAMP analysis of brain samples (for both experiments)
Each brain was extracted using 0.1 M HCl. 10 ml of 0.1 M HCl per g of tissue was added. Samples were sonicated then centrifuged at 10,000 rpm for 15 min at 4°C. The supernatent was poured off and used directly for measurement by ELISA. Dilutions were made using the 0.1 M HCl provided in the kit. Brain cAMP levels were measured using the direct cAMP ELISA kit (Enzo Life Sciences) according to the manufacturers instructions.
Experiment 2
We examined the concentration of Lixisenatide found in the brains of female WT mice following i.p. administration of 2.5, 25 or 250 nmol/kg bw of the peptide at 30 min and 3 h post injection. We also undertook a 3 week study on the effect of 25 nmol/kg bw daily administration of Lixisenatide on cell proliferation and new neuron development in the brains of WT mice.
Animals
In the chronic study, one group of female WT mice (n = 6) received once daily Lixisenatide (25 nmol/kg bw) for 3 weeks and the other group (n = 6) received saline (0.9% (w,v) NaCl) as control. At the end of the treatment period the mice were painlessly killed with an overdose of anaesthetic. Subsequently, animals were perfused with PBS, then with paraformaldehyde (PFA). Each brain was divided into 2 hemispheres, stored in PFA and kept at 4°C until further processing and analysis. In order to perform BrdU immunohistological analysis, animals were injected with 5-Bromo-2-DeoxyUridine (BrdU) (50 mg/kg) 24 h prior to sacrificing. Acid ethanol extraction of Lixisenatide from mice brains was performed as described above.
ELISA analysis of brain samples
Analysis of the Lixisenatide treated brains was carried out as recommended by the manufacturer of the total GLP-1 (7-36 and 9-36) ELISA kit, 43-GPTHU-E01, (Alpco. NH, USA). Briefly, standards, controls and test samples were added to the designated wells and then antibody mixture was added to each of these wells. The plate was sealed and incubated overnight at 4°C. Contents were then removed and the plate washed 5 times before adding HRP substrate. The plate was protected from exposure to light and incubated at RT for 20 min. Stop solution was then added, mixed gently and the absorbance read at 450 nm/620 nm within 10 min.
Cryostat preparation of brain tissue
One hemisphere from each animal was transferred from the PFA and placed in 30% sucrose solution overnight at 4°C. Each hemisphere was then mounted on the specimen disc using tissue-tek and Envi Ro Tech freeze spray. Brain sections were cut at 45 μm and placed in cryoprotect solution. Every sixth section was taken for analysis.
BrdU Immunohistochemical analysis
Cryoprotection solution was removed and sections washed in diH2O then incubated in H2O2. Sections were rinsed in H2O then incubated in 0.3% Triton X for 15 min. Following this sections were washed in PBS, incubated in HCl, then incubated in Borax, washed again in PBS then blocked in normal horse serum before incubating with the primary BrdU antibody (1:200) overnight at 4°C. On day 2 the sections were washed in PBS, incubated with the secondary antibody (anti-mouse, 1:200) for 30 min, washed in PBS, incubated in ABC reagent (Vector ABC Elite kit mouse) for 30 min, washed in PBS then SG substrate added until sufficient colour developed (2-10 min). Sections were then mounted on silanized slides. The total number of BrdU positive cells in the dentate gyrus were counted following stereological rules. Every 6th section was selected and 8 sections were stained for each animal. The average number per section was calculated. There were 6 animals in each treatment group. Therefore, the overall average per group was then calculated. The values are the average number of cells per animal. The doublecortin stained neurons were counted by the same method so they are also expressed as the average number per animal.
BrdU is a pyrimidine analogue of thymidine, it is selectively incorporated into the DNA of cells during the S phase of the cell cycle, therefore, it is an indicator of proliferating cells.
Double Cortin (DCX) immunohistological analysis
Excess cryoprotection solution was removed, sections washed in PBS, incubated in H2O2, washed in PBS, blocked in rabbit serum (5%) for 1 h then incubated in primary antibody (goat anti-DCX, 1:200) overnight at 4 C. The following day the sections were washed in PBS, incubated in secondary antibody (Vecta ABC kit anti-goat, 1:400) for 90 min, washed in PBS, incubated in ABC reagent for 90 min, washed in PBS, incubated in SG substrate until sufficient colour developed, then finally washed in diH2O before being mounted on salinized slides. DCX is a microtubule associated protein expressed almost exclusively in immature neurons. The number of new neurons observed on each section were counted and at least 8 sections were analysed per animal.
Statistical analysis
Results were analysed using Prism Graphpad software. Values are expressed as the mean ± SEM. Data was assessed using Students t-test and values were considered statistically significant if p < 0.05.
