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NEW FINDINGS What is the central question of this study? Angiotensin-(1-7) decreases cerebral infarct volume and improves neurological function when delivered centrally before and during ischaemic stroke. Here, we assessed the neuroprotective effects of angiotensin-(1-7) when delivered orally post-stroke. What is the main finding and its importance? We show that oral delivery of angiotensin-(1-7) attenuates cerebral damage induced by middle cerebral artery occlusion in rats, without affecting blood pressure or cerebral blood flow. Importantly, these treatments begin post-stroke at times coincident with the treatment window for tissue plasminogen activator, providing supporting evidence for clinical translation of this new therapeutic strategy. ABSTRACT As a target for stroke therapies, the angiotensin-converting enzyme 2-angiotensin-(1-7)-Mas [ACE2/Ang-(1-7)/Mas] axis of the renin-angiotensin system can be activated chronically to induce neuroprotective effects, in opposition to the deleterious effects of angiotensin II via its type 1 receptor. However, more clinically relevant treatment protocols with Ang-(1-7) that involve its systemic administration beginning after the onset of ischaemia have not been tested. In this study, we tested systemic post-stroke treatments using a molecule where Ang-(1-7) is included within hydroxypropyl-β-cyclodextrin [HPβCD-Ang-(1-7)] as an orally bioavailable treatment. In three separate protocols, HPβCD-Ang-(1-7) was administered orally to Sprague-Dawley rats after induction of ischaemic stroke by endothelin-1-induced middle cerebral artery occlusion: (i) to assess its effects on cerebral damage and behavioural deficits; (ii) to determine its effects on cardiovascular parameters; and (iii) to determine whether it altered cerebral blood flow. The results indicate that post-stroke oral administration of HPβCD-Ang-(1-7) resulted in 25% reductions in cerebral infarct volumes and improvement in neurological functions (P

Significant neuroprotective effects of angiotensin II type 2 (AT 2 ) receptor (AT 2 receptor) agonists in ischemic stroke have been previously demonstrated in multiple studies. However, the routes of agonist application used in these pre-clinical studies, direct intracerebroventricular (ICV) and systemic administration, are unsuitable for translation into humans; in the latter case because AT 2 receptor agonists are blood-brain barrier (BBB) impermeable. To circumvent this problem, in the current study we utilized the nose-to-brain (N2B) route of administration to bypass the BBB and deliver the selective AT 2 receptor agonist Compound 21 (C21) to näive rats or rats that had undergone endothelin 1 (ET-1)-induced ischemic stroke. The results obtained from the present study indicated that C21 applied N2B entered the cerebral cortex and striatum within 30 min in amounts that are therapeutically relevant (8.4-9 nM), regardless of whether BBB was intact or disintegrated. C21 was first applied N2B at 1.5 h after stroke indeed provided neuroprotection, as evidenced by a highly significant, 57% reduction in cerebral infarct size and significant improvements in Bederson and Garcia neurological scores. N2B-administered C21 did not affect blood pressure or heart rate. Thus, these data provide proof-of-principle for the idea that N2B application of an AT 2 receptor agonist can exert neuroprotective actions when administered following ischemic stroke. Since N2B delivery of other agents has been shown to be effective in certain human central nervous system diseases, the N2B application of AT 2 receptor agonists may become a viable mode of delivering these neuroprotective agents for human ischemic stroke patients.

Background: Compound 21 (C21), a selective small-molecule angiotensin type 2 receptor (AT2R) agonist, has been proven in multiple preclinical studies to reduce infarct size and ameliorate neurological deficits, when administered after ischemic stroke via intracerebroventricular or intraperitoneal routes. However, C21 poorly penetrates the blood brain barrier (BBB). In this study, we used the novel and non-invasive approach of intranasal trans-olfactory (INTO) application, in order to bypass the BBB and deliver C21 directly into the brain. The therapeutic efficacy of INTO application of C21 was assessed in a model of transient middle cerebral artery occlusion (MCAO). Methods: (i) Male SD rats (12 weeks old) underwent ischemic stroke by endothelin-1-induced MCAO. They were randomly divided into two treatment groups, either receiving 0.9% saline or C21 (1.5 ug/kg) at 1.5, 4, 24 and 48 h post-stroke, using a rat intranasal catheter device (Impel Neuropharma, Seattle, WA) for INTO application. All rats underwent blinded neurological assessments at 4, 24 and 72 h after stroke, and immediately after the 72 h tests, were euthanized and cerebral infarct volumes were assessed by TTC staining. (ii) Male SD rats (12 weeks old) underwent implantation of a telemetry transducer (DSI, St. Paul, MN) into the abdominal aorta for measurement of blood pressure, heart rate and locomotor activity after INTO C21 (1.5 ug/kg) vs. 0.9% saline at baseline and post-ischemic stroke. Results: (i) Post-stroke INTO delivery of C21 (1.5 ug/kg) elicited a significant lowering of % cerebral infarct volume (25.4 ± 4.7; n=9) compared with saline-treated rats (48.4 ± 4.4; n=21) [p Conclusions: Our results demonstrate, that INTO delivery of C21 exerts protective effects after ischemic stroke. These studies suggest INTO administration as potential future route of application of C21 to stroke patients.

Background: The renin angiotensin system is a promising target for stroke neuroprotection and therapy through activation of angiotensin type II receptors (AT2Rs). Compound 21 (C21), a selective non-peptide AT2R agonist, has been shown to exhibit neuroprotection and improve stroke outcomes in preclinical studies. Stimulation of AT2Rs is believed to counteract the negative effects of angiotensin type 1 receptor and provide distinctive beneficial anti-inflammatory and neurotropic effects. We hypothesized that C21 given after stroke through peripheral injections would have sustained neuroprotective effects in aged rats. Methods: Aged adult male SD rats (18-20 months) underwent ischemic stroke by monofilament middle cerebral artery occlusion (MCAO) and were randomly divided into two groups that received intraperitoneal (IP) injections of either 0.9% NaCl or 0.03mg/kg C21 at reperfusion (90 min), 24h, and 48h after stroke. All animals received blinded neurological exams at 4h, 24h, 72h, 7d, 14d, and 21d post-stroke. Infarct size was assessed by magnetic resonance imaging at 21 days. Results: Post-stroke treatment with C21 significantly improved neurological function, as evidenced by neurological testing using Rotarod and somatosensory dysfunction exams. At 7d and 14d after stroke, C21-treated rats had significantly increased Rotarod times versus saline-treated rats, and at 21d, the somatosensory function was significantly improved as measured by time to removal of paw adhesive. Infarct volume tended to be non-significantly decreased by C21 treatment at 21d post-stroke. Conclusions: Our findings indicate that targeting the renin-angiotensin system, specifically by stimulation of AT2Rs with C21, improves neurological function in aged rats with stroke over a sustained period of 21 days. These findings encourage further research into the renin-angiotensin system and specifically AT2Rs, and offers hope for effective alternatives for treating stroke.