Direct angiotensin AT2 receptor stimulation using a novel AT2 receptor agonist, compound 21, evokes neuroprotection in conscious hypertensive rats

Abstract

Background: In this study, the neuroprotective effect of a novel nonpeptide AT2R agonist, C21, was examined in a conscious model of stroke to verify a class effect of AT2R agonists as neuroprotective agents.

Methods and results: Spontaneously hypertensive rats (SHR) were pre-treated for 5 days prior to stroke with C21 alone or in combination with the AT2R antagonist PD123319. In a separate series of experiments C21 was administered in a series of 4 doses commencing 6 hours after stroke. A focal reperfusion model of ischemia was induced in conscious SHR by administering endothelin-1 to the middle cerebral artery (MCA). Motor coordination was assessed at 1 and 3 days after stroke and post mortem analyses of infarct volumes, microglia activation and neuronal survival were performed at 72 hours post MCA occlusion. When given prior to stroke, C21 dose dependently decreased infarct volume, which is consistent with the behavioural findings illustrating an improvement in motor deficit. During the pre-treatment protocol C21 was shown to enhance microglia activation, which are likely to be evoking protection by releasing brain derived neurotrophic factor. When drug administration was delayed until 6 hours after stroke, C21 still reduced brain injury.

Conclusion: These results indicate that centrally administered C21 confers neuroprotection against stroke damage. This benefit is likely to involve various mechanisms, including microglial activation of endogenous repair and enhanced cerebroperfusion. Thus, we have confirmed the neuroprotective effect of AT2R stimulation using a nonpeptide compound which highlights the clinical potential of the AT2R agonists for future development.

Figure 1. Infarct area when C21 is given as a pre-treatment.

Histological sections showing typical infarcted (darker area) and non-infarcted regions from SHR that were either treated with (A) vehicle, (B) AT2R agonist C21 50 ng/kg/min or (C) C21 50 ng/kg/min+PD123319 36 ng/kg/min. Mean data±SEM for infarct volume taken 72 hours post stroke in (D) cortical and (E) striatal regions on the ipsilateral side are shown for vehicle (n = 11), C21 at 5 ng/kg/min (n = 8), 10 ng/kg/min (n = 11) and 50 ng/kg/min (n = 11), AT₂R antagonist PD123319 alone (n = 10) and in combination with C21 50 ng/kg/min (n = 11). *P<0.05 vs. vehicle (1-way ANOVA).

Figure 2. Infarct area when C21-treatment is delay until 6 hours after stroke.

Histological sections showing typical infarcted (darker area) and non-infarcted regions from SHR that were either treated with (A) vehicle, (B) AT₂R agonist C21 144 µg/kg/dose or (C) C21 144 µg/kg/dose+AT₂R antagonist PD123319 103 µg/kg/dose. Mean data±SEM for infarct volume taken 72 hours post stroke in (D) cortical and (E) striatal regions on the ipsilateral side are shown for vehicle (n = 12), C21 (144 µg/kg/dose; n = 11), PD123319 alone (103 µg/kg/dose; n = 12) and in combination with C21 (144 µg/kg/dose; n = 6). *P<0.05 vs. vehicle (1-way ANOVA).

Figure 3. Motor deficit.

Motor deficit when C21 is given either pre-stroke (A) or post-stroke (B). For pre-treatment, the effect of vehicle (saline) control (n = 9), AT₂R agonist C21 at 5 ng/kg/min (n = 7), 10 ng/kg/min (n = 11) and 50 ng/kg/min (n = 11), AT₂R antagonist PD123319 alone (36 ng/kg/min; n = 10) and in combination with C21 (50 ng/kg/min; n = 8) on percentage errors made on the ledged beam following stroke. For post-stroke treatment, the effect of vehicle (n = 12), AT2R agonist C21 (144 µg/kg/dose; n = 11), AT2R antagonist PD123319 alone (103 µg/kg/dose; n = 12) and in combination with C21 (144 µg/kg/dose; n = 6) on percentage errors made on the ledged beam following stroke. Ledged beam test was performed pre-stroke (P) and at 1 (1 day) and 3 (3 day) post stroke. Mean data+SEM; ^*P<0.01 vs. vehicle pre-stroke (1-way RM ANOVA); ^#P<0.01 vs. corresponding time in vehicle-treated group (2-way RM ANOVA).

Figure 4. Neuronal survival.

Neuronal survival when C21 is given either pre-stroke (A) or post-stroke (B). For pre-treatment, the effect of vehicle (saline; n = 7); AT₂R agonist C21 alone (50 ng/kg/min; n = 8) and in combination with AT₂R antagonist PD123319 (36 ng/kg/min; n = 6) on neuronal survival at 72 hours post stroke. For post-stroke treatment, the effect of vehicle (n = 8), AT2R agonist C21 (144 µg/kg/dose; n = 8), AT2R antagonist PD123319 (103 µg/kg/dose; n = 8) in combination with C21 (144 µg/kg/dose; n = 6) on neuronal survival at 72 hours post stroke. Data expressed as the number of NeuN-immunopositive cells in infarcted (open columns) and non-infarcted (filled columns) cortical regions on the ipsilateral hemisphere. Mean data±SEM. *P<0.05 vs. corresponding region in vehicle group; #P<0.05 vs. non-infarcted region within same animal (1-way ANOVA).

Figure 5. Microglial activation.

Immunohistochemical sections from the cortex of the ipsilateral hemisphere from an animal receiving a pre-treatment of vehicle (A–C) or Compound 21 (50 ng/kg/min) depicting microglial activation using OX42 staining (green) in the non-infarcted tissue (A and D); the peri-infarcted tissue (B and E) or the core region of damage (C and F).

Figure 6. Number of activated microglial cells.

Number of activated microglial cells when C21 given either pre-stroke (A) or post-stroke. For pre-treatment, the effect of vehicle (saline; n = 6) and AT₂R agonist C21 (50 ng/kg/min; n = 5) on the number of activated microglia at 3 days after stroke. For post-stroke treatment, the effect of vehicle (saline; n = 7) and AT₂R agonist C21 (144 µg/kg/dose; n = 7) on the number of activated microglia at 3 days after stroke. Data expressed as number of activated microglia per 0.5 mm² area in non-infarcted (filled columns) and infarcted (open columns) region on the ipsilateral hemisphere. Mean data±SEM. **P<0.01 vs. corresponding region in vehicle-control (2-tailed unpaired t test).

Figure 7. BDNF-producing microglia are predominantly AT2R-positive.

Flow cytometry plots showing isolation of microglia (CD45^lo+OX42+ positive cells) and subsequently AT2R+ positive microglia that were also positive for BDNF (A). The mean±SEM frequency of AT2R expression within the population of microglia-producing BDNF obtained from SHR brains that had 3 days previously undergone either sham or stroke surgery (B). *P<0.05 vs. AT2R negative, BDNF-producing microglia (n = 4–9).

Figure 8. C21 elicits cerebral vasorelaxation through AT2R activation.

Cumulative concentration-response curves showing relaxation responses of isolated rat basilar arteries to C21 (10 nmolL –1 µmol/L) in the absence and presence of the AT2R antagonist PD123319 (10 µmol/L). Results are expressed as percent relaxation of U46619-induced tone and are given as mean±SEM (n = 5–7). *P<0.05 vs. control, (2-way ANOVA).