Centrally administered angiotensin-(1-7) increases the survival of stroke-prone spontaneously hypertensive rats

Abstract

What is the central question of this study? Activation of angiotensin-converting enzyme 2, resulting in production of angiotensin-(1-7) and stimulation of its receptor, Mas, exerts beneficial actions in a number cardiovascular diseases, including ischaemic stroke. A potential beneficial role for angiotensin-(1-7) in haemorrhagic stroke has not previously been reported. What is the main finding and its importance? Central administration of angiotensin-(1-7) into stroke-prone spontaneously hypertensive rats, a model of haemorrhagic stroke, increases lifespan and improves the neurological status of these rats, as well as decreasing microglial numbers in the striatum (implying attenuation of cerebral inflammation). These actions of angiotensin-(1-7) have not previously been reported and identify this peptide as a potential new therapeutic target in haemorrhagic stroke. Angiotensin-(1-7) [Ang-(1-7)] exerts cerebroprotective effects in ischaemic stroke, and this action is associated with a blunting of intracerebral inflammatory processes and microglial activation. Given that intracerebral inflammation and microglial activation play key roles in the mechanism of injury and brain damage in both ischaemic and haemorrhagic stroke, we have investigated the potential beneficial actions of Ang-(1-7) in stroke-prone spontaneously hypertensive rats (spSHRs), an established animal model of hypertension-induced haemorrhagic stroke. Angiotensin-(1-7) was administered by continuous infusion via the intracerebroventricular route for 6 weeks into spSHRs fed a high-sodium (4%) diet, starting at 49 days of age. This treatment resulted in a significant increase in survival of the spSHRs. Median survival was 108 days in control, artificial cerebrospinal fluid-infused spSHRs and 154 days in Ang-(1-7)-treated spSHRs. This effect was partly reversed by intracerebroventricular infusion of the Mas receptor blocker, A779. This Ang-(1-7) treatment also decreased the number of haemorrhages in the striatum, improved neurological status (reduced lethargy), decreased the number of microglia in the striatum and tended to increase neuron survival at the same site. Importantly, infusions of Ang-(1-7) had no effect on kidney pathology, heart pathology, body weight, serum corticosterone levels or blood pressure. This study is the first to demonstrate the cerebroprotective actions of Ang-(1-7), including increased survival time, in spSHRs. As such, these data reveal a potential therapeutic target for haemorrhagic stroke.

Conflict of interest statement

Conflict of Interest

None declared

Figures

Figure 1.. Central Ang-(1–7) administration improves the survival of spSHR.

Immediately following weaning at 35 days of age, spSHR were fed a 4% sodium diet. At 49 days of age, rats were infused ICV with either aCSF (0.15 μL/h), Ang-(1–7) [1.0 μg in 0.15 μl aCSF/h] or Ang-(1–7) + A779 (2.5 μg in 0.15 μl aCSF/h) for the next 6 weeks. Animals were monitored and maintained on the 4% sodium diet until death. Following death, brains were removed and the numbers and severity of hemorrhages in the cortex and subcortex/striatum were analyzed as described in the Methods. Panel (A) contains survival curves for spSHR following the above treatments. Vertical black dashed line indicates when the osmotic pumps stopped (91 days of age). N=9 rats for the aCSF and Ang-(1–7) groups; N=6 for the Ang-(1–7) + A779 group. *, P=0.0092, aCSF vs. Ang-(1–7) treatment; P=0.29, aCSF vs. Ang-(1–7) + A-779 treatment; P=0.07, Ang-(1–7) vs. Ang-(1–7) + A-779 treatment. Panels (B) and (D) show the numbers, and panels (C) and (E) the severity of hemorrhages in the cerebral cortex and subcortex/striatum, respectively. Bar graphs are means + SEM. *P<0.05, compared to spSHR infused with aCSF.

Figure 2.. Central Ang-(1–7) treatment improves the neurological status of spSHR.

Panel (A), Sunflower seed eating task: Rats were fed a 4% sodium diet and infused ICV with either aCSF (0.15 μL/h) or Ang-(1–7) [1.0 μg in 0.15 μl aCSF/h] as described in the legend to Figure 1, and underwent behavioral testing via the Sunflower seed-eating task every 3 days from 56 to 102 days of age. Vertical black dashed line indicates when the osmotic pumps stopped (91 days of age). Shown here is the number of shell pieces plotted against age. The timeline shows that the number of remaining shell pieces after eating 5 seeds is reduced by Ang-(1–7) treatment, as indicated by 2 way row matched ANOVA when comparing the lines. Data are means ± SEM. N=6 rats per group. Panels (B) and (C), Immobility and Rotation Frequency: Rats were subjected to the same treatments as in panel (A) and underwent testing for spontaneous locomotor activity between days 105–108 of age as described in the Methods. Bar graphs are means + SEM showing Immobility (B) and Rotation Frequency (C). N=9 rats for the aCSF treatment and 7 for Ang-(1–7). *P<0.05 compared to aCSF-treated spSHR.

Figure 3.. Central Ang-(1–7) infusion does not alter mean arterial blood pressure of spSHR.

Rats were fed a 4% sodium diet, infused ICV with either aCSF (0.15 μL/h) or Ang-(1–7) [1.0 μg in 0.15 μl aCSF/h] as described in the legend to Figure 1, and underwent measurements of MAP via tail cuff plethysmography every 2 to 5 days between days 70 and 110 of age. Vertical black dashed line indicates when the osmotic pumps stopped (91 days of age). Data are means ± SEM. N=8 rats per group.

Figure 4.. Anti-inflammatory action of centrally administered Ang-(1–7) in the striatum of spSHR.

(A-F) Rats were fed a 4% sodium diet, infused ICV with either aCSF (0.15 μL/h) or Ang-(1–7) [1.0 μg in 0.15 μl aCSF/h]. (A-F): At 90 days of age rats were deeply anesthetized, perfused transcardially with saline followed by 10% formalin and brains removed for immunostaining as described in the Methods. Panels A and B are representative fluorescence micrographs showing Iba-1 immunoreactivity in the striatum of aCSF and Ang-(1–7) infused rats, respectively. Panels C and D are representative fluorescence micrographs showing NeuN immunoreactivity in the striatum of aCSF and Ang-(1–7) infused rats, respectively. LV= lateral ventricle; cc = Corpus callosum. Stereological analyses of the striatum showing the estimated numbers of Iba-1 positive cells (microglia) and NeuN positive cells (neurons) are shown in panels E and F respectively. The bar graphs are means + SEM. N=5–6 rats/group. *P<0.05 compared to aCSF treated spSHR. (G, H): At 90 days of age, rats were euthanized, brains removed and processed for RT-PCR analyses of MCP-1 (G) and IL-1β (H) respectively. The bar graphs are means + SEM. N=5 rats/group.

Figure 5.. Co-localization of Mas with NeuN- and OX-42-immunopositive cells in the striatum.

The spSHRs were fed a 4% sodium diet and infused ICV with aCSF (0.15 μl h−1). At 90 days of age, rats were deeply anaesthetized, perfused transcardially with saline followed by 10% formalin and brains removed for immunostaining as described in the Methods. The representative fluorescence micrographs taken from the striatum at ×40 magnification are as follows: Mas (A); NeuN (B); co-localization of Mas and NeuN immunostaining (Mas+N; C); Mas (D); OX-42 (E); and Co-localization of Mas and OX-42 immunostaining (Mas+OX-42; F).

Figure 6.. Central Ang-(1–7) infusion does not reduce the pathological changes in kidney and heart structure that occur in spSHR.

Following euthanization of the 90- day-old rats used for analysis of microglial and neuronal numbers (Figure 4E,F), kidneys and hearts were removed and used to assess pathological changes. The bar graphs are means + SEM (N=5 rats/group) of scores obtained from the indicated kidney and heart pathological changes.