Stimulation of angiotensin II receptor 2 preserves cognitive function and is associated with an enhanced cerebral vascular density after stroke

Abstract

Angiotensin signaling is known to be sexually dimorphic. Although it is a well-studied target for intervention in stroke and cognitive impairment, female studies are rare. With females suffering a disproportionately greater negative impact of stroke and dementia vs. males, effective interventions are of utmost urgency. The aim of the current study was to determine the impact of activation of the angiotensin II type 2 receptor (AT2R) with the agonist compound 21 (C21) on the development of post-stroke cognitive impairment, after experimental ischemic stroke. Ovariectomized (OVX) spontaneously hypertensive rats (SHRs) were subjected to 1 h of middle cerebral artery occlusion (MCAO). At 24 h, rats with a significant neurologic deficit were randomized to receive either saline or C21 (0.03 mg/kg/day) intraperitoneally (IP) for 5 days, then orally (0.12 mg/kg/day) for a total of 6 weeks. Cognitive function, brain structure by MRI and vascular architecture by microCT angiography were measured. C21 preserved cognitive function, specifically spatial memory, and improved vascular density in the ischemic hemisphere at 6 weeks, reflecting both arteriogenesis and angiogenesis. In conclusion, C21 prevented cognitive impairment after stroke, likely through a mechanism involving vascular protection and restoration.

Keywords: Angiotensin II receptor subtype 2 (AT2R); Female rodents; Post-stroke cognitive impairment (PSCI); Spontaneously hypertensive rats (SHRs); microCT angiography.

Figure 1.

Experimental Design and Impact of stroke and C21 treatment on anxiety using the elevated plus maze. (a) Outline of the different time points at which behavioral and cognitive assessments were performed. EPM: elevated plus maze; PAT: passive avoidance test; OF: open field; NP: novel object placement test; MRI: magnetic resonance imaging. (b) Percent open arm entries of EPM. A main effect of disease was observed by 2-way ANOVA (p<0.05). (c) Percent time spent in open arms. A main effect of disease was observed by 2-way ANOVA (p<0.05). N= 6–9 per group. One animal spent the entire duration of the test in the closed arms and was excluded. Three animals jumped off the maze and were subsequently excluded from analysis.

Figure 2.

Impact of stroke and C21 treatment on memory. (a) Spatial reference memory was assessed using passive avoidance test. Baseline values of the latency to enter the shock arms were subtracted from test day values. Main effects of stroke was obtained by 2-way ANOVA (p<0.05). Post-hoc Bonferroni for multiple comparisons revealed a significant effect for stroke saline and C21 saline groups on latencies. # =compared to sham saline (p <0.05). $= compared to stroke saline (p<0.05). N=4–8 per group. (b) Impact of stroke and C21 treatment on short-term spatial memory using object placement test. A time course for object placement test performed using sham animals pre-stroke. Intertrial interval represents the time between the familiarization phase and test phase of OP task. Student t-test (p<0.05). (c) Percent exploration times in the OP task. A main effect of interaction between stroke and C21 treatment was observed by 2-way ANOVA (p<0.05). Bonferroni post-hoc test was carried out for multiple comparisons (p<0.05). #= compared to sham saline group and $= compared to stroked saline group.

Figure 3.

Impact of stroke and C21 treatment on brain structure using MRI. (a) Representative T2-weighted (T2W) coronal trans-axial MRI slices of different treatment groups. (b) Comparison of infarct size/cavitation between saline and C21-treated stroked animals, as a percent of the contralateral hemisphere. No difference was observed. Student t-test (p>0.05). (c) Ventricular volumes compared to contralateral volumes. (d) Ipsilateral hemisphere volume compared to total brain volume. (e) Total ventricular volumes compared to total brain volume. N=3–6 per group. A main effect of disease was observed by 2-way ANOVA (p<0.05). Post-hoc Bonferroni showed a significant effect for stroked saline group compared to sham saline group (p<0.05).

Figure 4.

Impact of Stroke and C21 treatment on cerebral architecture as viewed by micro-CT Angiography: (a) Brain region of interest studied by micro-CT angiography. The left and right hemispheres were studied. Regions highlighted by the red rectangle were studied using the circle of Willis and posterior cerebral arteries as landmarks. Analysis of blood vessels began at the start of the cerebral hemispheres and continued until the vessel mark by the green arrow. (b) 3D reconstructed images of all the stroked animals used in the study. (c) Histograms showing vessel separation (collateral formation) values from stroke side (SS) and non-stroked side (NS). (d) Histogram showing vessel thickness (lumen) values from stroke side and non-stroked side. Data are mean ± SD. N= 3–8 per group.

Figure 5.

Impact of Stroke and C21 treatment on vascular density and pro-anti-inflammatory cytokines at the ischemic border zone. (a) Representative photomicrographs of laminin immunofluorescence, in sham animals and at the ischemic border zone of stroked animals. Quantification of vessel number per field. Scale bar is 50 μm. n= 3–5 per group. (b) Representative Western blots of VEGF expression in the ipsilateral hemispheres of stroked and shams animals. N=3–5 per group.