Journal: Cell Reports

Article Title: Acute changes in systemic glycemia gate access and action of GLP-1R agonist on brain structures controlling energy homeostasis

doi: 10.1016/j.celrep.2022.111698

Figure Lengend Snippet: The access of Exendin-4 in the ARC under hypoglycemia involves tanycyte-borne VEGF (A) Quantification of hypothalamic Exendin-4_VT 750 (120 nmol/kg) fluorescent signal normalized to vehicle and (B) glycemic changes in response to VEGF (0.1 mg/kg, i.p.), insulin (240 nmol/kg, i.p. ) + VEGF, VEGF receptor antagonist Axitinib (25 mg/kg, i.p.), Axitinib alone, or the prostacyclin analog sodium beraprost (BPS 1 mg/kg, i.p.). n = 5–7/group. ∗ p < 0.05, vehicle versus insulin. Data are expressed as mean ± SEM. (C) Model for tanycyte-restricted invalidation of Vegfa in Tanycyte ΔVegfa mice (Vegfa lox/lox ; third ventricle injection of TAT-CRE or AAV-CRE-GFP) and experimental setup for concomitant injection of vehicle or 2-DG (250 mg/kg) together with Ex-4_Cy3 (120 nmol/kg) ∼15 min before sacrifice. (D and E) (D) Representative photomicrographs for Ex-4_Cy3 fluorescent distribution and (E) signal quantification in the dorsal (green circles) and ventral part (red circles) of the arcuate nucleus ∼15 min after 2-DG injection. Signal quantification was acquired on four to six brain sections from each animal, N = 2–5/group. Data are expressed as mean ± SEM. ∗ p < 0.05. (F) Glycemic change after saline (black, red) or insulin (0.75 U/kg, gray, orange) in control (black, gray) and Tanycyte ΔVegfa mice (red, orange). (G) 3D fluorescent signal quantification in the ARC in normoglycemic (NG) and hypoglycemic (HG) conditions. (H) Representative 2D planes from whole-brain light-sheet scanning to visualize fluorescent signal of peripherally injected Exendin-4_VT 750 (120 nmol/kg) in the ME/ARC region of control and Tanycyte ΔVegfa mice. (I) Signal quantification of fluorescent Exendin-4_VT 750 in the dorsal (green circles) and ventral part (red circles) of the arcuate nucleus 60 min after insulin (0.75 U/kg) injection. Signal quantification was acquired on three brain sections from each animal, n = 2–4/group. Data are expressed as mean ± SEM. ∗ p value < 0.05, insulin versus vehicle. For statistical details, see .

Article Snippet: Tanycytic specific knockdown of VEGFa was performed in isoflurane-anesthetized 8-weeks old Vegfa loxP/ loxP or tdTomato loxP−STOP-loxP Vegfa loxP/ loxP male mice by stereotactic injection of either TAT-Cre (Experimental group in C–4E) or AAV 1/2 -GFP (AAV 1/2 -CAG-eGFP; serotype 1:2 chimeric; titer = 1.2 x 10 13 GC/mL; Vector Biolabs) to produce control or AAV 1/2 -CRE-GFP (AAV 1/2 -CAG-iCre/eGFP; serotype 1:2 chimeric; titer = 2.8 x 10 13 ; Vector Biolabs) or AAV1/2 Dio2:Cre (serotype 1:2 chimeric, 0.5 × 10 10 genomic particles μl −1 , produce as previously described ( )) to produce Tanycyte ΔVegfa mice.

Techniques: Injection, Saline, Control

Journal: Cell Reports

Article Title: Acute changes in systemic glycemia gate access and action of GLP-1R agonist on brain structures controlling energy homeostasis

doi: 10.1016/j.celrep.2022.111698

Figure Lengend Snippet: Metabolic action of Exendin-4 involves tanycyte-borne VEGF (A) Experimental schedule for the characterization of metabolic efficiency in controls (Vegfa lox/lox ; ventricular injection of AAV-GFP) and Tanycyte ΔVegfa mice (Vegfa lox/lox ; ventricular injection of AAV-CRE-GFP) in response to daily i.p. saline injection (baseline, gray) followed by a 3-day treatment period consisting of a daily injection (2:00 p.m.) of Exendin-4 (120 nmol/kg, red), followed by insulin (20 nmol/kg, blue), and 3 days of mix of insulin + Exendin-4 (20 nmol/kg, 120 nmol/kg, green). Control and Tanycyte ΔVegfa mice were then exposed to a 3-week high-fat feeding regimen and reevaluated for their response to Exendin-4 or insulin + Exendin-4. Graphs represent averaged values for (B and C) cumulative food intake, (D and E) body weight change, (F and G) fat oxidation, and (H and I) food intake on chow diet. Three-day averaged cumulative food intake (J and K) and body weight change (L and M) through i.p. saline injection (black) followed by a 3-day treatment period consisting of a daily injection (2:00 p.m.) of Exendin-4 (120 nmol/kg, red) and 3 days of mix of insulin + Exendin-4 (20 nmol/kg, 120 nmol/kg, green) of control and Tanycyte ΔVegfa mice after exposure to high-fat diet. n = 8–5/group. Data are expressed as mean ± SEM. ∗ p < 0.05. $p < 0.05, insulin versus vehicle. £p < 0.05, Ex-4 versus vehicle. #p < 0.05, insulin + Ex-4 versus vehicle. &p < 0.05, insulin + Ex-4 versus Ex-4. For statistical details, see .

Article Snippet: Tanycytic specific knockdown of VEGFa was performed in isoflurane-anesthetized 8-weeks old Vegfa loxP/ loxP or tdTomato loxP−STOP-loxP Vegfa loxP/ loxP male mice by stereotactic injection of either TAT-Cre (Experimental group in C–4E) or AAV 1/2 -GFP (AAV 1/2 -CAG-eGFP; serotype 1:2 chimeric; titer = 1.2 x 10 13 GC/mL; Vector Biolabs) to produce control or AAV 1/2 -CRE-GFP (AAV 1/2 -CAG-iCre/eGFP; serotype 1:2 chimeric; titer = 2.8 x 10 13 ; Vector Biolabs) or AAV1/2 Dio2:Cre (serotype 1:2 chimeric, 0.5 × 10 10 genomic particles μl −1 , produce as previously described ( )) to produce Tanycyte ΔVegfa mice.

Techniques: Injection, Saline, Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: iPSC-hepatocyte organoids as a novel platform to predict AAV gene therapy efficacy

doi: 10.1016/j.omtm.2025.101467

Figure Lengend Snippet: Analysis of the AAV transduction in iPSC-derived hepatocyte organoids (A) AAV transduction efficiency with different concentrations (MOI 10 4 , 10 5 , and 10 6 ) for donor one and two for 9 days. Data are expressed as mean (SD) ( n = 3). (B) Phase-contrast and immunofluorescence images of AAV5 and AAV8 (MOI 10 6 ) transduction iPSC-derived hepatocyte organoids at day 2, day 14, and day 28. Scale bar, 500 μm. (C) AAV transduction efficiency at a 10 6 MOI overtime in percentage for the organoids (4 donors) for 28 days. Data are expressed as mean (SD) ( n = 3). (D) Heatmap representing the mean of the percentage of GFP-positive organoids 28 days after AAV treatment (MOI 10 6 ) for the four-organoid donors for AAV1 to AAV9.

Article Snippet: AAV constructs produced in Sf9 cells through infection with two recombinant baculoviruses were sourced from Virovek: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, and AAV9 expressing green fluorescent protein (GFP) and empty AAV2, AAV8, and AAV9 were used under a cytomegalovirus (CMV) promoter.

Techniques: Transduction, Derivative Assay, Immunofluorescence