The Non-peptide Angiotensin-(1–7) Mimic AVE 0991 Attenuates Delayed Neurocognitive Recovery After Laparotomy by Reducing Neuroinflammation and Restoring Blood-Brain Barrier Integrity in Aged Rats

Figures

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  Figure 1. Effects of laparotomy on serum and hippocampal RAS activity in aged rats. Eighteen-month-old rats received laparotomy under sevoflurane anesthesia. Laparotomy significantly increased RAS activity in the hippocampus, leading to a decrease in Ang-(1–7) levels (A) and an increase in Ang II levels (C) but no significant changes were detected in serum RAS activity (B,D). Values are the mean ± SEM (n = 8 per group). *p < 0.05, vs. control group. RAS, renin-angiotensin system; Ang, angiotensin.

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  Figure 2. Correlations between Ang II and Ang-(1–7) levels after surgical trauma. Eighteen-month-old rats received laparotomy under sevoflurane anesthesia. Levels of Ang II and Ang-(1–7) in hippocampal tissues and blood serum were dynamically determined at 12, 24, and 48 h after surgery using ELISA. Correlation coefficients (r) and P-values were calculated using Spearman’s test. (A,C) In the hippocampus, the Ang II level was negatively correlated with that of Ang-(1–7). (B,D) Nevertheless, there was no significant correlation between levels of Ang II and Ang-(1–7) in the circulation. Values are the mean ± SEM (n = 8 per group). Ang, angiotensin; ELISA, enzyme-linked immunosorbent assay.

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  Figure 3. Laparotomy decreased hippocampal Mas receptor expression. Eighteen-month-old rats received laparotomy under sevoflurane anesthesia in the presence or absence of AVE 0991 treatment. Mas protein expression was determined at 24 h after surgery. Representative western blot bands (A) and statistical analysis (B) of Mas receptor expression levels. Values are the mean ± SEM (n = 4 per group). *p < 0.05, vs. control group; #p < 0.05, vs. surgery group.

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  Figure 4. AVE 0991 treatment alleviated the hippocampal-dependent spatial memory impairment in aged rats after surgery. (A) Escape latency for rats to reach the platform in the navigation trials, which measures the ability of the animals to acquire spatial information. (B) Swimming speeds of the aged rats. (C) Percentage of exploration time spent within the target quadrant during probe trials. (D) Percentage of exploration distance within the target quadrant during probe trials. (E) A representative exploration trajectory. Values are the mean ± SEM (n = 10 per group). *p < 0.05, vs. control group; #p > 0.05, vs. surgery group.

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  Figure 5. AVE 0991 treatment restored the surgery-induced imbalance in MMP-9/TIMP-3 in the aged hippocampus. Eighteen-month-old rats received laparotomy under sevoflurane anesthesia in the presence or absence of AVE 0991 treatment. Immediately after the surgical challenge, AVE 0991 was intranasally administered at a dose of 0.9 mg/kg. Representative western blot bands (A) and statistical analysis (B–D) of MMP-9, TIMP-3, and the ratio of MMP-9/TIMP-3. (E,F) Immunofluorescence results for MMP-9 and TIMP-3 in the hippocampal CA1 subregion at 6 h after surgery. Magnification, ×200. Scale bar, 50 μm. Values are the mean ± SEM (n = 4 per group). *p < 0.05, vs. control group; #p < 0.05, vs. surgery group. N.S., no significance; MMP-9, matrix metalloproteinase-9; TIMP-3, tissue inhibitor of matrix metalloproteinase-3.

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  Figure 6. AVE 0991 treatment alleviated surgery-induced neuroinflammation in the aged hippocampus. Eighteen-month-old rats received laparotomy under sevoflurane anesthesia in the presence or absence of AVE 0991 treatment. Representative western blot bands (A) and statistical analysis (B–F) CD11b, an indicator of microglial activation, and inflammation-related molecules, including TNF-α, IL-1β, HMGB1, and the HMGB1 receptor RAGE. Values are the mean ± SEM (n = 4 per group). *p < 0.05, vs. control group; #p < 0.05; vs. surgery group. CD11b, cluster of differentiation 11b; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; HMGB1, high mobility group box-1; RAGE, receptor for advanced glycation end products.

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  Figure 7. AVE 0991 treatment attenuated the surgery-induced BBB permeability. Eighteen-month-old rats received laparotomy under sevoflurane anesthesia in the presence or absence of AVE 0991 treatment. (A–E) The expression of the TJ proteins ZO-1, occludin, claudin-5 in the hippocampus was determined by western blot analysis. Representative western blot bands (A) and statistical analysis (B–D) of ZO-1, occludin, claudin-5. (E) Endogenous IgG immunohistochemical staining in the hippocampal CA1 subregion was examined (magnification × 200, scale bar, 50 μm). (F) The results of the semi-quantification of IgG extravasation were shown. Values are the mean ± SEM (n = 4 per group). *p < 0.05, vs. control group; #p < 0.05, vs. surgery group. N.S., no significance; BBB, blood-brain barrier; TJ, tight junction; ZO-1, zonula occludens-1.

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  Figure 8. Mas activation with intranasal AVE 0991 treatment attenuated surgery-induced disruption of BBB ultrastructure. Eighteen-month-old rats received laparotomy in the presence or absence of AVE 0991 treatment. Disruption of BBB ultrastructure was assessed by transmission electron microscopy (TEM) of a coronal section through the hippocampal CA1 subfield (n = 4 per group). At 24 h postsurgery, the basal laminas had partly collapsed (red arrowhead) and the ultrastructural integrity of BBB appeared destroyed, the angioedema was evident, astrocytic end-feet (*, red asterisk) were swollen, the perivascular spaces around the basal laminas were enlarged. After AVE 0991 treatment, the pathological changes in BBB ultrastructure were alleviated, shown by a clear recovery of the basal laminas and diminished angioedema surrounding the capillaries. Scale bar, 1 μm. L, vascular cavity. BBB, the blood-brain barrier.

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  Figure 9. Schematic illustration of the probable mechanisms underlying delayed neurocognitive recovery (dNCR) in aged rats after anesthesia and surgery. Surgical stress activates the central RAS in the aged hippocampus, and the resultant shift in the balance of the central Ang II/Ang-(1–7) leads to NF-κB-mediated neuroinflammation and an imbalance of MMP/TIMP. This further causes BBB disruption and dNCR following surgery. Targeting the Ang II/Ang-(1–7) imbalance using ARBs (Li et al., 2016) and/or the Ang-(1–7) mimic AVE 0991 may be promising therapeutic strategies for combating postoperative neurocognitive impairment through an anti-neuroinflammatory manner and restoring the BBB integrity. dNCR, delayed neurocognitive recovery; RAS, renin-angiotensin system; Ang, angiotensin; NF-κB, nuclear factor-κB; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase; BBB, blood-brain barrier; ARBs, angiotensin receptor blockers.