Figure 1.
Experimental design and Aβ intraventricular injection effect on survival.
A, Experimental procedures time-line, (I) at 1 hpf embryos were placed in 6-well plates and exposed to LiCl 100 µM or H2O; (II) at 24 hpf embryos were removed from their chorion, and injected with Aβ1-42 10 µM or its vehicle; LiCl and H2O solutions were replaced daily throughout the experiment; (III) at 5dpf larvae behavior were evaluated and samples for protein and gene expression quantification were obtained. B, Representative image of Aβ (red) injected on the brain ventricle area. C, Kaplan-Meier survival comparison for all groups throughout the experiment showed significant effects (Log-rank (Mantel-Cox) test, p = 0.0415, N = 60 in triplicates) that were not statistically significant when individual comparisons were performed.
Figure 2.
Intraventricular Aβ injection significantly impairs avoidance of an aversive stimulus.
5dpf larvae escape behavior from an aversive stimulus (charts were plotted with means and SD escape responses to a non-stimuli area). Two-way ANOVA followed by Bonferroni demonstrated a significant effect of treatment factor (H2O and LiCl) (p<0.0001; F(1,166) = 40.77; N = 10 in triplicates). Aβ injected animals showed diminished escape responses when compared to their vehicle control group in H2O and LiCl-treated groups (* indicates p<0.05 for both comparisons). LiCl treatment increased escape responses in all groups when compared to their respective H2O-treated equivalent (a indicates p<0.05 for noninjected Ø groups; p<0.0001 for veh-injected groups and p<0.001 for Aβ-injected groups in Student-t test.
Figure 3.
Intraventricular Aβ injection increases tau-p at Ser202 and Thr205 residues and this effect is reversed by lithium treatment. Representative Western blots showing immunoreactivity to phosphorylated tau protein normalized to β-actin and quantification of absorbance (charts were plotted with means and SD). Two-way ANOVA followed by Bonferroni demonstrated a significant effect of treatment factor (p<0.0001, F(1,42) = 296.02; N = 3 in triplicates). H2O–Aβ injected animals showed increased levels of tau phosphorylation in relation to H2O-veh (*p<0.001). LiCl treatment decreased tau-p in all groups when compared to their respective H2O-treated equivalent (a indicates p<0.0001 in Student-t test for all comparisons).
Figure 4.
Intraventricular injection alters apoptotic targets.
A, representative Western blots showing immunoreactivity of indicated proteins normalized to β-actin. B, Western blots quantification of absorbance (charts were plotted with means and SD). Two-way ANOVA followed by Bonferroni posttest didn’t show significant differences (p = 0.1153, F(2,41) = 2.28 for p53; p = 0.3063, F(2,48) = 1.21 for bax; p = 0.4420, F(2,45) = 0.83 for caspase-8; N = 3 in triplicates) in Aβ injected animals compared to their vehicle control group in H2O or LiCl-treated groups. P53 and caspase-8 levels differed between H2O-veh and H2O–Aβ and noninjected H2O–Ø controls (*p<0.05, **p<0.01). Among noninjected animals, LiCl treatment increased p53 and caspase-8 protein levels compared to their respective H2O-treated equivalent (a indicates p<0.01 for caspase-8 and p<0.0001 for p53 in Student-t test). C, q-PCR analysis normalized to three constitutive genes (b-actin, rpl13a and ef1a) (charts were plotted with means and SD). Two-way ANOVA followed by Bonferroni posttest didn’t show significant differences on gene expression (p = 0.5473, F(2,88) = 0.61 for p53; p = 0.7313, F(2,48) = 0.31 for bax; p = 0.8822, F(2,50) = 0.13 for bcl-2; N = 6 in duplicates).