Angiotensin-(1-7) improves cognitive function and reduces inflammation in mice following mild traumatic brain injury

Abstract

Introduction: Traumatic brain injury (TBI) is a leading cause of disability in the US. Angiotensin 1-7 (Ang-1-7), an endogenous peptide, acts at the G protein coupled MAS1 receptors (MASR) to inhibit inflammatory mediators and decrease reactive oxygen species within the CNS. Few studies have identified whether Ang-(1-7) decreases cognitive impairment following closed TBI. This study examined the therapeutic effect of Ang-(1-7) on secondary injury observed in a murine model of mild TBI (mTBI) in a closed skull, single injury model.

Materials and methods: Male mice (n = 108) underwent a closed skull, controlled cortical impact injury. Two hours after injury, mice were administered either Ang-(1-7) (n = 12) or vehicle (n = 12), continuing through day 5 post-TBI, and tested for cognitive impairment on days 1-5 and 18. pTau, Tau, GFAP, and serum cytokines were measured at multiple time points. Animals were observed daily for cognition and motor coordination via novel object recognition. Brain sections were stained and evaluated for neuronal injury.

Results: Administration of Ang-(1-7) daily for 5 days post-mTBI significantly increased cognitive function as compared to saline control-treated animals. Cortical and hippocampal structures showed less damage in the presence of Ang-(1-7), while Ang-(1-7) administration significantly changed the expression of pTau and GFAP in cortical and hippocampal regions as compared to control.

Discussion: These are among the first studies to demonstrate that sustained administration of Ang-(1-7) following a closed-skull, single impact mTBI significantly improves neurologic outcomes, potentially offering a novel therapeutic modality for the prevention of long-term CNS impairment following such injuries.

Keywords: Mas receptor; angiotensin 1-7; cognitive impairment; pTau; traumatic brain injury.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Bruhns, Sulaiman, Gaub, Bae, Davidson Knapp, Larson, Smith, Coleman, Staatz, Sandweiss, Joseph, Hay, Largent-Milnes and Vanderah.

Figures

Figure 1

Closed skull traumatic brain injury (TBI) induced variable cognitive outcomes as determined by novel object recognition (NOR). (A) Number of animals grouped by 20% bins showing both cognitive decline and cognitive improvement. Differences in (B) hit velocity, (C) time to inspire, and (D) time to ambulate port impact were not different between mice with cognitive impairment or normal cognition. (E,F) Animals with a mild TBI (<20% decline or impairment) performed similar to uninjured animals in NOR, whereas moderate and severe outcomes were statistically different from baseline. Statistically significant difference (**p < 0.01, ***P < 0.001, and ****P < 0.0001), n = 55 mice.

Figure 2

Cognitive deficits after closed-skull TBI are mitigated by Ang-(1-7). (A) Diagram of Novel Object Recognition set up. (B) Discrimination Ratio as a function of time demonstrates relative maintenance of cognitive function in mice treated with Ang-(1-7) as opposed to normal saline alone. Time of TBI infliction is denoted by the vertical dashed line. Primary administration of either drug or saline vehicle took place 2 h post-TBI. All subsequent drug and saline injections took place 30 min prior to NOR assays conducted on days 2–16. repeated measures ANOVA (with Tukey correction) was employed to analyze main effect of the drug intervention on DR, followed by Tukey Range test where appropriate. Twenty-four male mice 12 mice in each group, statistically significant (*p < 0.05) difference between treatment groups; Statistically significant (‡p < 0.05) difference from baseline within the group.

Figure 3

A779 MASR antagonist pre-treatment attenuated Ang-(1-7)-mediated cognitive improvement. (A) The NOR discrimination index (DI) was statistically lower in A779 (1 mg/kg, i.p.)-pretreated animals vs. saline on days 2 and 4 of treatment post TBI (*p < 0.05). (B) This was confirmed after calculation of the Area under the curve (AUC) to account for differences over time. (C,D) Showed no differences in total distance moved or speed were observed (P > 0.05). Repeated measures ANOVA (with Tukey correction) was employed. N = 12 mice in each group. **P < 0.01, ns; no significant.

Figure 4

Histology of cerebral cortex after mTBI and Ang1-7 intervention (A) HandE staining of cortex for naïve, saline (N = 4), and Ang-(1-7) (N = 4) treated groups days 1, 3, 7, 14 post-TBI. (B) Mouse picture gives location of the single mTBI to the closed skull just above the left parietal lobe. Ang-(1-7) (1 mg/kg, i.p./day on days 1–5) treated mice have significantly higher cortical cell count compared to the saline-treated mice (days 1–5, i.p. p < 0.03). (C,D) No significant difference was found for cortical thickness and corpus callosum thickness between Ang-(1-7) (1 mg/kg, i.p./day on days 1–5) treated mice and saline treated mice.

Figure 5

Histology of hippocampus after mTBI and Ang1-7 intervention (A) HandE staining of left (ipsilateral) hippocampus and right (contralateral) hippocampus for naïve, saline (N = 4), and Ang-(1-7) (N = 4) (1 mg/kg, i.p./day on days 1–5) treated groups on days 1, 3, 7, and 14 post-TBI. (B) Left hippocampus shows Ang-(1-7) treated mice maintain higher hippocampal cell counts in the CA3 region compared to the saline treated mice on days 1, 3, 7, 14 post-TBI. (C) Right hippocampus shows Ang-(1-7) treated mice maintain higher hippocampal cell counts in the CA3 region compared to saline treated mice on days 1 and 3 post-TBI.

Figure 6

Protein changes in ipsilateral cortex following TBI (N = 3–4 mice/group). (A) Pooled protein densities of cortex from Western blot studies demonstrate a significant decrease for pTau on days 1, 3, 7, and 14 after a mTBI in both Ang-(1-7) (1 mg/kg, i.p. days 1–5) and saline treated animals. Yet, a significant difference was seen on days 3, 7, and 14 in Ang-(1-7) treated animals as compared to saline with less pTau expression. (B) There was a significant increase in Tau on day 7 but not on days 1, 3, or 14, Ang-(1-7) did have a significant effect in increasing overall Tau on day 7 but was no different to saline on the other days. (C) The pTau:Tau ratio is expressed relative to 1.0, the pre-TBI control value and demonstrated a significant difference on days 3, 7, and 14 between saline and Ang-(1-7) treatment. (D) GFAP was represented as Percent Control Expression for post-TBI days 1, 3, 7, 14. GFAP expression in cortex demonstrated a reverse-U-shaped curve with levels increasing on days 1 and 14 but decreasing on days 3 and 7. Ang-(1-7) demonstrated a significant decrease in GFAP on days 1 and 14 as compared to saline treatment. NC (normal control) is pre-TBI control protein levels standardized to a value of 100. “°” indicates a statistically significant difference (p < 0.05) in expression level or ratio between the selected post-TBI day and baseline for saline treated animals. “Δ” indicates a statistically significant difference (p < 0.05) in expression level or ratio between the selected post-TBI day and baseline for Ang-(1-7) treated animals. “*” indicates a statistically significant difference (p < 0.05) in expression level or ratio between the Ang-(1-7) and Saline treated groups for the selected post-TBI day (N = 4 mice/group).

Figure 7

Protein changes in hippocampus following TBI (n = 3–4 mice/group). (A) Pooled protein densities of left hippocampus from Western blot studies demonstrate a significant decrease for pTau on days 1, 3, 7, and 14 after a mTBI in saline treated animals. Yet, a significant difference was seen on days 1 and 7 in Ang-(1-7) (1 mg/kg, i.p. days 1–5) treated animals as compared to saline with less pTau expression. There were no significant differences on days 3 and 14 between saline and Ang-(1-7) treatments. (B) There was a significant increase in Tau on days 3, 7, and 14 after the mTBI in the Ang-(1-7) treatment with day 14 doubling in the amount of hippocampal Tau protein due to Ang-(1-7) treatment. (C) The pTau:Tau ratio demonstrated a significant difference on days 1 and 7 with Ang-(1-7) demonstrating a significant decrease in pTau as compared to saline treated animals. A similar trend was seen on days 3 and 14. (D) GFAP was represented as Percent Control Expression for post-TBI days 1, 3, 7, and 14. GFAP expression in cortex demonstrated an increase in levels from baseline on days 3, 7, and 14 with no significant difference between treatment groups. NC (normal control) is pre-TBI control protein levels standardized to a value of 100. “°” indicates a statistically significant difference (p < 0.05) in expression level or ratio between the selected post-TBI day and baseline for saline treated animals. “Δ” indicates a statistically significant difference (p < 0.05) in expression level or ratio between the selected post-TBI day and baseline for Ang-(1-7) treated animals. * indicates a statistically significant difference (p < 0.05) in expression level or ratio between the Ang-(1-7) and Saline treated groups. *P < 0.05.

Figure 8

Qualitative changes in serum cytokine levels (n = 3–4 mice/group). (A) CXCL13 expression increases after 3 days post-mTBI in saline treated animals. Ang-(1-7) (1 mg/kg, i.p.) significantly normalized this mTBI induced CXCL13 increase on D3 (*p < 0.05). No other increases or decreases in CXCL13 expression were observed over the observation period of 14 d. (B) M-CSF expression decreases below naïve levels after day 3 post-mTBI in saline and Ang-(1-7) treated animals, at 7 d (p < 0.05) with no difference between saline and Ang-(1-7) groups. (C) TIMP-1 expression significantly increases in Ang-(1-7) treated animals 1-day post TBI as compared to naïve controls; this increase was not observed in saline treated animals until D3 post TBI 3d post-mTBI; Ang-(1-7) treated animals expressed TIMP-1 at significantly lower levels than saline treated animals (p = 0.03).