Cerebroprotection by angiotensin-(1-7) in endothelin-1-induced ischaemic stroke

Abstract

Activation of angiotensin-converting enzyme 2 (ACE2), production of angiotensin-(1-7) [Ang-(1-7)] and stimulation of the Ang-(1-7) receptor Mas exert beneficial actions in various peripheral cardiovascular diseases, largely through opposition of the deleterious effects of angiotensin II via its type 1 receptor. Here we considered the possibility that Ang-(1-7) may exert beneficial effects against CNS damage and neurological deficits produced by cerebral ischaemic stroke. We determined the effects of central administration of Ang-(1-7) or pharmacological activation of ACE2 on the cerebral damage and behavioural deficits elicited by endothelin-1 (ET-1)-induced middle cerebral artery occlusion (MCAO), a model of cerebral ischaemia. The results of the present study demonstrated that intracerebroventricular infusion of either Ang-(1-7) or an ACE2 activator, diminazine aceturate (DIZE), prior to and following ET-1-induced MCAO significantly attenuated the cerebral infarct size and neurological deficits measured 72 h after the insult. These beneficial actions of Ang-(1-7) and DIZE were reversed by co-intracerebroventricular administration of the Mas receptor inhibitor, A-779. Neither the Ang-(1-7) nor the DIZE treatments altered the reduction in cerebral blood flow elicited by ET-1. Lastly, intracerebroventricular administration of Ang-(1-7) significantly reduced the increase in inducible nitric oxide synthase mRNA expression within the cerebral infarct that occurs following ET-1-induced MCAO. This is the first demonstration of cerebroprotective properties of the ACE2-Ang-(1-7)-Mas axis during ischaemic stroke, and suggests that the mechanism of the Ang-(1-7) protective action includes blunting of inducible nitric oxide synthase expression.

Figure 1. Effects of angiotensin-(1–7) [Ang-(1–7)] on endothelin-1 (ET-1)-induced middle cerebral artery (MCA) constriction and cerebral blood flow

A–I are photomicrographs showing the MCA branches (arrows) during 80 μM ET-1-induced vasoconstriction. Primary and secondary branches of the MCA were visualized with a surgical microscope after temporal craniotomy to create a cranial window. Images were captured at a rate of 1 min⁻¹, starting immediately prior to ET-1 injection (0 min), throughout the ET-1 injection (3 min) and for at least 60 min after initiation of the ET-1 injection. Representative images are shown for rats that underwent 7 days of intracerebroventricular (I.C.V.) artificial cerebrospinal fluid (aCSF) pretreatment (0.5 μl h⁻¹) prior to a sham middle cerebral artery occlusion [MCAO; 3 μl of 0.9% saline (Sal) injection, A–C], 7 days of I.C.V. aCSF pretreatment (0.5 μl h⁻¹) prior to an ET-1-induced MCAO (3 μl of 80 μM ET-1 injection, D–F), or 7 days of I.C.V. Ang-(1–7) (1.1 nM; 0.5 μl h⁻¹) pretreatment prior to an ET-1-induced MCAO (G–I). J shows vasoconstriction of primary or secondary MCA branches quantified as the percentage of baseline vessel diameter. Data are means ± SEM from four rats per treatment condition. Baseline vessel diameter was determined prior to ET-1 or 0.9% saline injection (time 0 min). *P < 0.05 for aCSF versus sham MCAO and †P < 0.05 for Ang-(1–7) versus sham MCAO [two-way repeated-measures ANOVA (P < 0.001) followed by Bonferroni post hoc test]. K shows cerebral blood flow (CBF) in the ischaemic core and penumbra during ET-1-induced (MCAO). Laser Doppler flowmetry was used to investigate the CBF reduction that results in cortical areas both adjacent (ischaemic core) and distal (ischaemic penumbra) to the site of ET-1 (80 μM) injection. Representative traces from one rat are shown for each probe position. The data are presented as a percentage of the baseline signal. L shows the effects of I.C.V. infusion of Ang-(1–7) on CBF in the vascular territory of the MCA distal to the site of ET-1 injection. Data are means ± SEM of the percentage change from baseline CBF. Endothelin-1 injection took place over a period of 3 min starting at 0 min on this graph. No significant differences exist between Ang-(1–7) (n = 6) and aCSF (n = 6) treatment groups at any time point (two-way repeated-measures ANOVA).

Figure 2. Intracerebral pretreatment with Ang-(1–7) reduces CNS infarct size after ET-1-induced MCAO

Rats were pretreated via the I.C.V. route with Ang-(1–7) (1.1 nM; 0.5 μl h⁻¹; n = 9), aCSF (n = 23), Ang-(1–7) + A-779 (1.14 nM; 0.5 μl h⁻¹; n = 9) or A-779 (n = 7) alone for 7 days prior to MCAO induced by intracranial injection of ET-1 as described in the Methods. In addition, two rats received a sham MCAO with 0.9% saline injection in place of ET-1. Brains were removed for TTC staining 72 h after the ET-1-induced MCAO. A, representative brain sections showing infarcted (white) and non-infarcted grey matter (red) in the treatment conditions indicated. B, bar graphs are means + SEM showing the percentage infarcted grey matter in each treatment group. *P < 0.05 versus aCSF; †P < 0.05 versus Ang-(1–7). C, D and E are representative fluorescence micrographs showing NeuN immunoreactivity co-localized with DAPI nuclear stain (pink coloured cells; red NeuN + blue DAPI) in the cerebral cortex of rats that underwent sham stroke surgery, ET-1-induced MCAO + I.C.V. infusion of aCSF (0.5 μl h⁻¹; 7 days) or ET-1-induced MCAO + I.C.V. infusion of Ang-(1–7) (1.1 nM; 0.5 μl h⁻¹; 7 days).

Figure 3. Intracerebral pretreatment with Ang-(1–7) reduces behavioural deficits 72 h after ET-1-induced MCAO

Rats were pretreated via the I.C.V. route with Ang-(1–7) (1.1 nM; 0.5 μl h⁻¹; n = 9), aCSF (n = 23), Ang-(1–7) + A-779 (1.14 nM; 0.5 μl h⁻¹; n = 9) or A-779 (n = 7) alone for 7 days prior to ET-1-induced MCAO, as described in the Methods. The bar graphs shown here are the means ± SEM of data obtained from the Bederson neurological examination (A) and the Garcia neurological examination (B), as well as the sunflower seed-eating test for the time to eat five seeds (C) and the number of shell pieces (D). Also shown in each panel are data from two rats that underwent I.C.V. infusion of aCSF as above and a sham stroke (0.9% saline injection instead of ET-1). *P < 0.01 versus aCSF/ET-1 group; †P < 0.01 versus Ang-(1–7) group.

Figure 4. Intracerebral pretreatment with diminazine aceturate (DIZE) reduces CNS infarct size and behavioural deficits after ET-1-induced MCAO

Rats were pretreated via the I.C.V. route with water (DIZE solvent; n = 15), DIZE (5 μg (0.5 μl)⁻¹ h⁻¹); n = 18), DIZE + A-779 (1.14 nM; 0.5 μl h⁻¹; n = 7) or A-779 (n = 10) alone for 7 days prior to MCAO induced by intracranial injection of ET-1 (80 μM). A, brains were removed for TTC staining 72 h after stroke. Representative brain sections (top) show infarcted (white) and non-infarcted grey matter (red) in the above-described treatment conditions. Bar graphs (bottom) are means + SEM showing the percentage infarcted grey matter in each treatment group. *P < 0.001 versus water. B, bar graphs are means + SEM showing the Bederson examination scores in each treatment condition, 72 h following the ET-1-induced MCAO. *P < 0.05 versus water. C, effects of I.C.V. infusion of DIZE (5 μg (0.5 μl)⁻¹ h⁻¹) on CBF in the vascular territory of the MCA distal to the site of ET-1 injection. Data are means ± SEM of the percentage change from baseline CBF. Endothelin-1 injection took place over a period of 3 min starting at 0 min. No significant differences exist between DIZE (n = 6) and water (n = 6) treatment groups at any time point (two-way repeated-measures ANOVA). D, effects of I.C.V. infusion of DIZE (5 μg (0.5 μl)⁻¹ h⁻¹) on systolic blood pressure. Bar graphs are means + SEM (n = 6 rats per group). *P < 0.01 versus water.

Figure 5. Intracerebral gene expression elicited by ET-1-induced MCAO: effects of Ang-(1–7)

A–E, rats were pretreated via I.C.V. infusion with either aCSF (0.5 μl h⁻¹) or Ang-(1–7) (1.1 nM; 0.5 μl h⁻¹) for 7 days prior to ET-1-induced MCAO or sham stroke (injection of 0.9% saline instead of ET-1). Twenty-four hours later, rats were killed, after which brains were removed and processed for qRT-PCR as detailed in the Methods. The bar graphs are means + SEM of Mas, AT1R, ACE2 iNOS, and IL-6 mRNA levels from the whole ipsilateral (right) hemisphere of each treatment group. *P < 0.05 versus corresponding sham treatment (n = 5–6 rats per group). F, rats were pretreated exactly as described for A–E. The bar graphs are means + SEM of iNOS mRNA levels from the ipsilateral (right) cerebral cortex stroke zone of each treatment group. *P < 0.05 versus corresponding sham treatment; †P < 0.05 versus corresponding ET-1/aCSF group; (n = 5–8 rats per group). Note that these data are presented as (ΔΔCt) values, thus a lower value represents an increase in mRNA level.