Deficiency of Endothelial Nitric Oxide Synthase (eNOS) Exacerbates Brain Damage and Cognitive Deficit in A Mouse Model of Vascular Dementia

Abstract

Vascular Dementia (VaD) accounts for nearly 20% of all cases of dementia. eNOS plays an important role in neurovascular remodeling, anti-inflammation, and cognitive functional recovery after stroke. In this study, we investigated whether eNOS regulates brain damage, cognitive function in mouse model of bilateral common carotid artery stenosis (BCAS) induced VaD. Late-adult (6-8 months) C57BL/6J and eNOS knockout (eNOS-/-) mice were subjected to BCAS (n=12/group) or sham group (n=8/group). BCAS was performed by applying microcoils to both common carotid arteries. Cerebral blood flow (CBF) and blood pressure were measured. A battery of cognitive functional tests was performed, and mice were sacrificed 30 days after BCAS. Compared to corresponding sham mice, BCAS in wild-type (WT) and eNOS-/- mice significantly: 1) induces short term, long term memory loss, spatial learning and memory deficits; 2) decreases CBF, increases ischemic cell damage, including apoptosis, white matter (WM) and axonal damage; 3) increases blood brain barrier (BBB) leakage, decreases aquaporin-4 (AQP4) expression and vessel density; 4) increases microglial, astrocyte activation and oxidative stress in the brain; 5) increases inflammatory factor interleukin-1 receptor-associated kinase-1(IRAK-1) and amyloid beta (Aβ) expression in brain; 6) increases IL-6 and IRAK4 expression in brain. eNOS-/-sham mice exhibit increased blood pressure, decreased iNOS and nNOS in brain compared to WT-sham mice. Compared to WT-BCAS mice, eNOS-/-BCAS mice exhibit worse vascular and WM/axonal damage, increased BBB leakage and inflammatory response, increased cognitive deficit, decreased iNOS, nNOS in brain. eNOS deficit exacerbates BCAS induced brain damage and cognitive deficit.

Keywords: Vascular dementia; bilateral common carotid artery stenosis (BCAS); endothelial nitric oxide synthase (eNOS); white matter injury.

Figure 1.

BCAS significantly induces cognitive dysfunction compared to WT-sham mice. eNOS-/-BCAS mice exhibit severe cognitive function impairment compared to eNOS-/-sham or WT-BCAS mice, respectively. (A) Water Maze test; Latency: *p<0.05. (B) Odor Test; (C) Novel Object Test.

Figure 2.

BCAS significantly decreases CBF compared to WT-sham or eNOS-/-sham control mice, respectively. eNOS-/-BCAS mice exhibit significantly decreased CBF at 1 and 14 days after BCAS compared to WT-BCAS mice. eNOS-/-sham and eNOS-/-BCAS significantly increases SAP, DAP and MAP compared to WT-sham or WT-BCAS mice, respectively. (A) CBF measurement and qualification; (B) DAP, SAP and MAP measurement and quantification data. *p<0.05, n=8/group.

Figure 3.

BCAS significantly increases brain lesion volume, apoptosis and WM/axonal damage compared to WT-sham mice. eNOS-/-BCAS mice exhibit significantly increased lesion volume, apoptosis and WM/axonal damage compared to eNOS-/-sham or WT-BCAS mice, respectively. (A) HE staining and quantification data; (B) Tunel staining and quantification data; (C) LFB and BS staining and quantification data. *p<0.05; HE staining: n=8/group; Tunel: n=8/group; LFB and BS: n=6/group.

Figure 4.

BCAS significantly induces BBB leakage and decreases AQP4 expression and vessel density compared to WT-sham mice. eNOS-/-BCAS exhibits severe BBB leakage and decreased vessel density compared to eNOS-/-sham or WT-BCAS mice, respectively. eNOS-/-BCAS significantly decreased AQP4 expression compared to eNOS-/-sham control mice. (A) Albumin; (B) AQP-4; (C) vWF staining and quantification data; *p<0.05, n=8/group.

Figure 5.

BCAS significantly increases microglial and astrocyte expression, NOX-2, IRAK1 and Aβ expression in brain compared to WT-sham mice. eNOS deficit significantly increases microglial and astrocyte activity, NOX-2, IRAK1 and Aβ expression in brain compared to WT-sham control mice; eNOS-/-BCAS exhibits increased expression of microglial, astrocyte, NOX-2, Aβ and IRAK1 expression compared to eNOS-/-sham or WT-BCAS mice, respectively. (A) IBA-1; (B) GFAP; (C) NOX-2; (D) Amyloid-β; (E) IRAK1. *p<0.05, n=8/group.

Figure 6.

BCAS significantly increases IL-6 and IRAK4 compared to WT-sham mice. eNOS-/-BCAS mice shows decreased iNOS and nNOS expression compared to WT-BCAS or eNOS-/-sham mice. (A) iNOS and nNOS; (B) IL-6; (C) IRAK4 gene expression. n=6/group; *p<0.05.