Role of angiotensin system modulation on progression of cognitive impairment and brain MRI changes in aged hypertensive animals - A randomized double- blind pre-clinical study

Abstract

Growing evidence suggests that renin angiotensin system (RAS) modulators support cognitive function in various animal models. However, little is known about their long-term effects on the brain structure in aged hypertensive animals with chronic cerebral hypoperfusion as well as which specific domains of cognition are most affected. Therefore, in the current study we examined the effects of Candesartan and Compound 21 (C21) (RAS modulators) on aspects of cognition known to diminish with advanced age and accelerate with hypertension and vascular disease. Outcome measures for sensorimotor and cognitive function were performed using a sequence of tests, all blindly conducted and assessed at baseline and after 4 and 8 weeks of chronic hypoxic hypoperfusion and treatment. Magnetic resonance imaging (MRI) was performed at the end of the 8 week study period followed by animal sacrifice and tissue collection. Both Candesartan and C21 effectively preserved cognitive function and prevented progression of vascular cognitive impairment (VCI) but only candesartan prevented loss of brain volume in aged hypertensive animals. Collectively, our findings demonstrate that delayed administration of RAS modulators effectively preserve cognitive function and prevent the development / progression of VCI in aged hypertensive animals with chronic cerebral hypoperfusion.

Keywords: AT2 Receptor; Angiotensin type 1 receptor blockade; Hypertension; MRI; Rat; Renin-angiotensin; Vascular cognitive impairment.

Fig. 1

Mechanism of cognitive dysfunction induced by chronic hypertension and cerebral hypoperfusion/vascular insufficiency

Fig. 2

Schematic depiction of the experimental design

Fig. 3. Effect of mild cerebral hypoperfusion on the fluid intake and body weight of aged hypertensive animals

The (A) fluid intake and (B) weight trend of aged SHRs, at various time points, before and after UCCAO (n=8–10 animals/group). Symbols and error bars indicate mean and SEM, compound symmetric variance co-variance structure provided the best model fit. Differences between groups over time were examined using the estimated least squares means from a repeated measures mixed model and using a Bonferroni adjustment to the overall alpha level for the number of comparisons made between groups, any differences were nonsignificant.

Fig. 4. Effect of mild cerebral hypoperfusion on the motor function of aged hypertensive animals

Swim speeds on the initial and reversal training/test of the Morris Water Maze test were taken as measures of swimming ability, a parameter of motor function (n = 8–9 animals/group). Error bars indicate SEM, Repeated measures ANOVA mixed models were used to examine differences in outcomes, for the initial training, between the groups over time. Statistical significance for post hoc comparisons using Bonferroni adjustment to the overall alpha level for the number of post hoc pair-wise comparisons, *P<0.0056, the Bonferroni adjusted alpha. For the reversal training and testing outcomes, a one-way ANOVA was used to examine differences between the groups and a Tukey-Kramer multiple comparison test was used to examine post hoc pair-wise differences*P<0.05. Any differences were nonsignificant.

Fig. 5. Effect of RAS modulator treatment on the non-spatial working memory of aged hypertensive animals with mild cerebral hypoperfusion

Effect of treatment on the (A) Discrimination Index (DI) and (B) Recognition Index (RI) of aged hypertensive animals, at different time points after ischemic insult (n=8–10 animals/group). Symbols and error bars indicate mean and SEM. Repeated measures ANOVA, mixed models, were used to examine differences in outcomes, between the three groups over time with post hoc pair-wise comparisons denoted by *P<0.0042, the Bonferroni adjusted alpha.

Fig. 6. Effect of RAS modulator treatment on spatial reference memory of aged hypertensive animals with mild cerebral hypoperfusion

Spatial reference memory evaluated with the Morris Water Maze probe test in aged SHRs. The platform was removed and rats were allowed to swim for 60 s in an attempt to find it. Performance was evaluated by measuring (A) initial latency to the target zone as well as (B) time spent in the target zones. (C) Heat maps illustrating relative time spent in the various locations during the probe tests both before (pre-probe) as well as 4 weeks after (pre-probe) after initial ischemic procedure. The target (NE) quadrant is indicated with black lines (n=8–10 animals/group). Symbols and error bars indicate mean and SEM. Repeated measures ANOVA, mixed models, were used to examine differences in outcomes between the three groups over time with post hoc pair-wise comparisons denoted by *P<0.0056, the Bonferroni adjusted alpha.

Fig. 7. Effect of RAS modulator treatment on cognitive flexibility/new learning in aged hypertensive animals with mild cerebral hypoperfusion

Cognitive flexibility and “new” learning was assessed by the reversal training/test. Performance was evaluated by measuring (A) Latency to target quadrant (B) Latency to target/platform zone (C) Distance to platform zone, which is how close they swim to target location while attempting to find it (D) Duration in target zone (E) Duration in previous quadrant, which indicates preservative behavior in aged hypertensive animals and (F) Heat maps illustrating relative time spent in the various locations during the reversal, the target (SE) quadrants is indicated with black lines (n = 8–10 animals/group). Bars and error bars indicate mean and SEM. A one-way ANOVA was used to examine differences between the groups and a Tukey-Kramer multiple comparison test was used to examine post hoc pair-wise differences. Statistical significance for post hoc comparisons using Tukey’s multiple comparison procedure are denoted by *P<0.05.

Fig. 7. Effect of RAS modulator treatment on cognitive flexibility/new learning in aged hypertensive animals with mild cerebral hypoperfusion

Cognitive flexibility and “new” learning was assessed by the reversal training/test. Performance was evaluated by measuring (A) Latency to target quadrant (B) Latency to target/platform zone (C) Distance to platform zone, which is how close they swim to target location while attempting to find it (D) Duration in target zone (E) Duration in previous quadrant, which indicates preservative behavior in aged hypertensive animals and (F) Heat maps illustrating relative time spent in the various locations during the reversal, the target (SE) quadrants is indicated with black lines (n = 8–10 animals/group). Bars and error bars indicate mean and SEM. A one-way ANOVA was used to examine differences between the groups and a Tukey-Kramer multiple comparison test was used to examine post hoc pair-wise differences. Statistical significance for post hoc comparisons using Tukey’s multiple comparison procedure are denoted by *P<0.05.

Fig. 8. Effect of RAS modulator treatment on brain structure in aged hypertensive animals with mild cerebral hypoperfusion using Magnetic Resonance Imaging (MRI)

Representative T2-weighted (T2W) (A) coronal trans-axial MRI slices and (B) axial whole brain images of the different treatment groups compared to young normotensive wistar control (C) Coronal trans-axial FLAIR images of the 3 treatment groups. Relative measures of brain damage/atrophy (D) Total brain volume (E) Hippocampal volume (F) Ventricular volume (H) Representative trans-axial FLAIR images (G) White matter hyperintensities (WMH). The yellow arrows indicate those affecting the periventricular white matter tracks; the red arrows indicate those affecting the lateral septal nucleus in addition to the dorsal and intermediate portions. (n =3–5 animals/group). Error bars indicate SEM. A one-way ANOVA was used to examine differences between the groups and a Tukey-Kramer multiple comparison test was used to examine post hoc pair-wise differences. Statistical significance for post hoc comparisons using Tukey’s multiple comparison procedure are denoted by *P < 0.05.

Fig. 8. Effect of RAS modulator treatment on brain structure in aged hypertensive animals with mild cerebral hypoperfusion using Magnetic Resonance Imaging (MRI)

Representative T2-weighted (T2W) (A) coronal trans-axial MRI slices and (B) axial whole brain images of the different treatment groups compared to young normotensive wistar control (C) Coronal trans-axial FLAIR images of the 3 treatment groups. Relative measures of brain damage/atrophy (D) Total brain volume (E) Hippocampal volume (F) Ventricular volume (H) Representative trans-axial FLAIR images (G) White matter hyperintensities (WMH). The yellow arrows indicate those affecting the periventricular white matter tracks; the red arrows indicate those affecting the lateral septal nucleus in addition to the dorsal and intermediate portions. (n =3–5 animals/group). Error bars indicate SEM. A one-way ANOVA was used to examine differences between the groups and a Tukey-Kramer multiple comparison test was used to examine post hoc pair-wise differences. Statistical significance for post hoc comparisons using Tukey’s multiple comparison procedure are denoted by *P < 0.05.

Fig. 8. Effect of RAS modulator treatment on brain structure in aged hypertensive animals with mild cerebral hypoperfusion using Magnetic Resonance Imaging (MRI)

Representative T2-weighted (T2W) (A) coronal trans-axial MRI slices and (B) axial whole brain images of the different treatment groups compared to young normotensive wistar control (C) Coronal trans-axial FLAIR images of the 3 treatment groups. Relative measures of brain damage/atrophy (D) Total brain volume (E) Hippocampal volume (F) Ventricular volume (H) Representative trans-axial FLAIR images (G) White matter hyperintensities (WMH). The yellow arrows indicate those affecting the periventricular white matter tracks; the red arrows indicate those affecting the lateral septal nucleus in addition to the dorsal and intermediate portions. (n =3–5 animals/group). Error bars indicate SEM. A one-way ANOVA was used to examine differences between the groups and a Tukey-Kramer multiple comparison test was used to examine post hoc pair-wise differences. Statistical significance for post hoc comparisons using Tukey’s multiple comparison procedure are denoted by *P < 0.05.

Fig. 9. Effect of RAS modulator treatment on Aβ_1-42 concentrations in the pre-frontal cortex of aged hypertensive animals with mild cerebral hypoperfusion

Quantitative determination of Aβ_1-42 concentrations in cortical lysates of aged hypertensive animals, comparing the different treatment groups at 8 weeks post-UCCAO (n = 8–9 animals/group). Error bars indicate SEM. A one-way ANOVA was used to examine differences between the groups and a Tukey-Kramer multiple comparison test was used to examine post hoc pair-wise differences. Statistical significance for post hoc comparisons using Tukey’s multiple comparison procedure are denoted by *P<0.05.