Fig. 4: Hypothalamic insulin resistance, but not hypothalamic inflammation and microglial activation, persist beyond acute stages of infection in mice.

A Representative image of immunofluorescence labeling for ZIKV (NS2B protein, green) in ZIKV infected mice at 30 dpi. Scale bar = 200 μm, inset scale bar = 50 μm. B–D Bar graphs represent NPY (B; n = 5/group, Student’s t-test t = 0.8756, p = 0.4068), AgRP (C; n = 7 mock, 8 ZIKV, Student’s t-test t = 0.4565, p = 0.6556) and POMC (D; n = 6 mock, 7 ZIKV, Student’s t-test t = 1.020, p = 0.3296) mRNA levels measured in the hypothalamus of mock-infused or ZIKV-infected mice at 30 dpi. Data are expressed as mean ± SEM. E Heat Map generated using relative gene expression of TNFα (n = 9/group, Student’s t-test t = 0.2754, p = 0.7866), IL-6 (n = 5 mock, 4 ZIKV, Student’s t-test t = 0.1850, p = 0.8593), IL-1β (n = 9 mock, 8 ZIKV, Student’s t-test t = 1.752, p = 0.1001), pan IFNA (n = 4 mock, 3 ZIKV, Student’s t-test t = 0.5184, p = 0.6943), IFNβ (n = 5/group, Student’s t -test t = 0.3830, p = 0.717), IFNg (n = 4 mock, 3 ZIKV, Student’s t-test t = 0.3642, p = 0.7306), ISG15 (n = 4 mock, 3 ZIKV, Student’s t-test t = 0.2735, p = 0.7954) in the hypothalamus of mice at 30 dpi. F, G Representative images of Iba immunoreactivity (green) in the lateral region of the hypothalamus of mock-infused (F) or ZIKV-infected mice (G) at 30 dpi. Scale bar, 25 μm; inset scale bar, 10 μm. F’, G’ Individual cells highlighted in dashed-line rectangles in (F, G) were transformed into binary composites and are shown on the right of each representative image. H–J Bar graphs represent the number of Iba-1+ cells (H; n = 4/group, Student’s t-test, t = 1.050, p = 0.3340), Feret’s diameter of Iba-1+ cells (I; n = 4/group, 10-15 cells per mouse, Student’s t-test, t = 1.513, p = 0.1811), and the percentage of Iba-1+ cells with a resting or activated phenotype (J; n = 4/group, Student’s t-test) in the hypothalamus of mock-infused or ZIKV-infected mice 30 dpi. K IR mRNA levels in the hypothalamus of mice at 30 dpi (Student’s t test, t = 0.7381, p = 0.4718). L, M Representative Western Blot image (L) and quantification (M) of IR (n = 6/group, Student’s t-test, t = 3.293, *p = 0.0081) protein levels in the hypothalamus of mock-infused and ZIKV-infected mice at 30 dpi; GAPDH was used as a loading control. Representative images from Western blot experiments were always run on the same gels but sometimes in noncontiguous lanes. N, O For determination of insulin-induced anorexic effect, mice were injected i.c.v. with mock medium or ZIKV and after 30 days received 200 mU of insulin or saline as control. Food intake was measured in the metabolic cage after 4 h (O; n = 9 mock+Veh, mock+insulin, ZIKV + Veh, ZIKV + Insulin, *p = 0,0986, one-way ANOVA followed by Tukey’s post hoc). Data are representative of two independent experiments with similar results. Data are expressed as mean ± SEM. Each symbol represents a different experimental subject.