Gradual hypertension induction in middle‐aged Cyp1a1‐Ren2 transgenic rats produces significant impairments in spatial learning

Figure 1. I3C treatment significantly increases blood pressure. I3C treatment significantly increases both (A) systolic and (B) diastolic blood pressure. Indirect blood pressures were measured using the tail‐cuff method approximately once each week. I3C‐treated animals (n = 13) demonstrated a time dependent increase in blood pressure throughout the experimental period. Small filled circles are the measured values for individual animals in each group (dark gray: I3C treated, light gray: Control, n = 12). Diamonds are the group means and the error bars are the standard deviation of the means. Linear model fit lines are included. These results demonstrate that the I3C treatment was sufficient to increase blood pressure in the Cyp1a1‐Ren2 animals. Two‐way repeated measures ANOVA was performed and the post hoc P‐value for Experimental Day is indicated at the top of the plot (*P < 0.05, **P < 0.01, ***P < 0.0001, N.S. = not significant, P > 0.05).

Figure 2. Hypertension reduced spatial memory performance on the Morris water maze. Spatial memory as assessed by Morris water maze performance was measured before (PRE) and after (POST) long‐term treatment with I3C (n = 13) or Control (n = 12) chow (dark gray: I3C treated, light gray: Control). Animals were randomly assigned to treatment groups; however, we noted a trend in the control chow‐assigned animals to perform worse during the PRE phase. Thus we calculated a Cognitive Savings score for each individual animal (the difference in performance between the PRE and POST tasks). We used a mixed effect model with animal ID as a random effects variable and both test day and group (treatment vs. control group) as fixed effects variables. Treatment affected the Cognitive Savings score (χ 2(1) = 4.11, P = 0.04), with treated animals experiencing less savings 0.17 ± 0.079 (SEM) than controls. These results demonstrate that the I3C treatment impaired spatial reference memory performance in the Cyp1a1‐Ren2 animals.

Figure 3. Hypertension has no effect on visual cued Morris swim task performance. A visual cued version of the Morris swim task was performed during the PRE and POST phases of the experiment (dark gray: I3C treated, n = 13; light gray: Control, n = 12). There were no statistically significant differences detected due to I3C treatment. These results demonstrate that hypertension did not significantly impair vision or alter swimming speed in the Cyp1a1‐Ren2 animals.

Figure 4. I3C treatment significantly increases cardiac and renal measurements of end organ damage. End organ damage was measured in each animal at sacrifice. I3C (n = 13) treated animals are shown here as dark gray, and control (n = 12) animals as light gray. These results demonstrate that the I3C induction of hypertension was sufficient to increase common measures of cardiac and renal end organ damage in the Cyp1a1‐Ren2 animals. Wilcoxon ranked sum test P‐values are indicated for each comparison.

Figure 5. Hypertension results in significantly elevated cardiac and renal fibrosis. Representative images from heart and kidney tissue using Masson's trichrome stain to indicate collagen fibrosis. Images that scored nearest the group means are illustrated here.

Figure 6. Hypertension induces end organ damage in the aorta. I3C (n = 13) treatment induced aortic hypertrophy as evidenced by increases in medial cross‐sectional area (CSA), medial thickness, and the media to lumen ratio. Increased cellular proliferation was evaluated based on the number of cells positively labeled with anti‐proliferating cell nuclear antigen (PCNA). Wilcoxon ranked sum test P‐values are indicated for each comparison.

Figure 7. I3C treatment is associated with significant body weight loss after 2 weeks. The I3C (n = 13) treated animals have significant drops in body weight by the end of the experimental period. Post hoc analysis suggests that weight tends to diverge by day 14 (**P < 0.01, ***P < 0.001, N.S. = not significant).

Figure 8. Weight loss results in no significant changes in spatial or cued behavior, and treatment did not affect blood pressure. The food restricted group (n = 3) did show significant weight loss by two‐way repeated measures ANOVA, (F [28,140] = 24.90, P < 0.001). Post hoc analysis revealed a significance difference at day 27 (*), P < 0.001). No significant differences in systolic or diastolic blood pressures were observed between groups, nor were there differences in the spatial or cued versions of the Morris swim task (P > 0.05).