Angiotensin receptor (AT2R) agonist C21 prevents cognitive decline after permanent stroke in aged animals-A randomized double- blind pre-clinical study

Abstract

Post stroke cognitive impairment (PSCI) is an understudied, long-term complication of stroke, impacting nearly 30-40% of all stroke survivors. No cure is available once the cognitive deterioration manifests. To our knowledge, this is the first study to investigate the long-term effects of C21 treatment on the development of PSCI in aged animals. Treatments with C21 or vehicle were administered orally, 24 h post-stroke, and continued for 30 days. Outcome measures for sensorimotor and cognitive function were performed using a sequence of tests, all blindly conducted and assessed at baseline as well as at different time points post-stroke. Our findings demonstrate that the angiotensin receptor (AT2R) agonist C21 effectively prevents the development of PSCI in aged animals.

Keywords: AT2 receptor; Compound 21; Permanent stroke; Post-stroke cognitive impairment; RAS modulation.

Fig. 1:

Schematic depiction of experimental design

Fig. 2:. Effect of AT2R stimulation on the body weight of aged wistar animals post-stroke.

The weight trend is represented as % of baseline (pre-surgical weight). A compound symmetric variance co-variance structure provided the best model fit. Differences between groups for specific days (0, 1, 8, 10, and 30) 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 (Group x Day x Day effect: F=3.92, P=0.0895. (n = 6-7 animals/group). Symbols and error bars indicate mean and SEM, *indicate significantly different from sham # indicate significantly different from treatment

Fig. 3:. Effect of AT2R stimulation on the sensorimotor function of aged wistar animals post-stroke.

Sensorimotor function in aged animals determined by (A) Bederson test of gait and motor coordination (Group × Time interaction: F_(1,∞)=0.50, P<0.4776) and (B) Swim speeds on both initial and reversal tests of the Morris Water Maze (Probe Test F_(1,12,.6)=4.53, p=0.0536; Reversal Test t₍₁₂₎=−0.55, p=0.5949) as measures of swimming ability (a parameter of motor function) for the different treatment groups, (n = 7 animal s/group). Error bars indicate SEM,

Fig. 4:. Effect of AT2R stimulation on the non-spatial working memory of aged wistar animals post-stroke.

Effect of treatment on the (A) Discrimination Index (DI) (Group x Time interaction: F_(2.16)=23.33, P<0.0001) and (B) Recognition Index (RI) (Group × Time interaction: F_(2.16)=54.33, P=0.0003) of aged wistar rats, at 21 days post-stroke (n = 7 animals/group). Repeated measures ANOVA mixed models were used to examine differences in outcomes, between the groups over time. Bars and error bars indicate mean and SEM. Statistical significance is denoted by *P<0.001 for post hoc pair-wise comparisons from baseline and #P<0.001 for post hoc pair-wise comparisons “between groups” post-stoke, using the Bonferroni adjusted alpha.

Fig. 5:. Effect of AT2R stimulation on reference memory and associative learning in aged wistar animals post-stroke.

Reference memory and associative learning was assessed by the (A) Passive avoidance test (Group × Time interaction: F_(2.16)=10.73, P<.0000l) and Morris Water Maze probe test in aged wistar rats. The platform was removed and rats were allowed to swim for 60 s in an attempt to find it. Performance was evaluated by measuring (B) initial latency to the target zone (Group×Time interaction: F_(1,8.22)=4.26, p=0.0719) as well as (C) time spent in the target zones (GroupxTime interaction: F_(1,4)=3.86, p=0.0695). (D) Heat maps illustrating relative time spent in the various locations during the probe tests both before (pre-op probe) as well as 4 weeks after stroke (post-op probe). The target (NE) quadrant is indicated with black lines. (E) Initial training latency to target quadrant (Group ×Time interaction: F_(1,13.22)=4.25, p=0.0596). (n = 7 animals/treatment group) Symbols and error bars indicate mean and SEM, Repeated measures ANOVA mixed models were used to examine differences in outcomes, between the groups over time.

Fig. 6:. Effect of AT2R stimulation on cognitive flexibility/ new learning in aged wistar animals post-stroke.

Cognitive flexibility and “new” learning was assessed by the reversal training/ test. Performance was evaluated by measuring (A) Latency to target zone (t₍₁₂₎=−2.77, p=0.0169) (B) Distance to platform zone, which is how close they swim to target location while attempting to find it(t₍₁₂₎=−3.47, p=0.0046), (C) Duration in new target (SE) quadrant (t₍₁₂₎=3.31, p=0.0062) and (D) platform zone (t₍₁₂₎=2.69, p=0.0198) which indicate effective consolidation and retention of the new memory (E) Heat maps illustrating relative time spent in the various locations during the reversal, the target (SE) quadrants is indicated with black lines. The (F) Latency to previous target (t₍₁₂₎=l.99, p=0.0830), (G) Duration in previous (NE) target quadrant (t₍₁₂₎=−1.60, p=0.1353) (H) Frequency to previous target (t₍₁₂₎=−2.71, p=0.0189) are parameters indicating preservative behavior.(n = 7 animals/group, bars and error bars indicate mean and SEM, a one-way ANOVA or two-sample t-test were used to examine differences between the groups and a Tukey-Kramer multiple comparison test was used to examine post hoc pair-wise differences.

Fig. 6:. Effect of AT2R stimulation on cognitive flexibility/ new learning in aged wistar animals post-stroke.

Cognitive flexibility and “new” learning was assessed by the reversal training/ test. Performance was evaluated by measuring (A) Latency to target zone (t₍₁₂₎=−2.77, p=0.0169) (B) Distance to platform zone, which is how close they swim to target location while attempting to find it(t₍₁₂₎=−3.47, p=0.0046), (C) Duration in new target (SE) quadrant (t₍₁₂₎=3.31, p=0.0062) and (D) platform zone (t₍₁₂₎=2.69, p=0.0198) which indicate effective consolidation and retention of the new memory (E) Heat maps illustrating relative time spent in the various locations during the reversal, the target (SE) quadrants is indicated with black lines. The (F) Latency to previous target (t₍₁₂₎=l.99, p=0.0830), (G) Duration in previous (NE) target quadrant (t₍₁₂₎=−1.60, p=0.1353) (H) Frequency to previous target (t₍₁₂₎=−2.71, p=0.0189) are parameters indicating preservative behavior.(n = 7 animals/group, bars and error bars indicate mean and SEM, a one-way ANOVA or two-sample t-test were used to examine differences between the groups and a Tukey-Kramer multiple comparison test was used to examine post hoc pair-wise differences.

Fig. 7:. Effect of AT2R stimulation on Aβ_1-42 and BDNF in concentrations in the pre-frontal cortex of aged wistar animals post-stroke.

Quantitative determinations of (A) Aβ_1-42 and (B) BDNF concentrations in the pre-frontal cortical lysates of aged wistar rats at 30 days post-stroke, (n = 7 animals/group, Error bars indicate SEM, Statistical significance for post hoc comparisons using Tukey’s multiple comparison procedure are denoted by *P < 0.05)