Noninvasive Vagus Nerve Stimulation Prevents Ruptures and Improves Outcomes in a Model of Intracranial Aneurysm in Mice

Abstract

Background and Purpose- Inflammation is a critical determinant of aneurysmal wall destabilization, growth, and rupture risk. Targeting inflammation may suppress aneurysm rupture. Vagus nerve stimulation (VNS) has been shown to suppress inflammation both systemically and in the central nervous system. Therefore, we tested the effect of a novel noninvasive transcutaneous VNS approach on aneurysm rupture and outcome in a mouse model of intracranial aneurysm formation with wall inflammation. Methods- Aneurysms were induced by a single stereotaxic injection of elastase into the cerebrospinal fluid at the skull base, combined with systemic deoxycorticosterone-salt hypertension, without or with high-salt diet, for mild or severe outcomes, respectively. Cervical VNS (two 2-minute stimulations 5 minutes apart) was delivered once a day starting from the day after elastase injection for the duration of follow-up. Transcutaneous stimulation of the femoral nerve (FNS) served as control. Multiple aneurysms developed in the circle of Willis and its major branches, resulting in spontaneous ruptures and subarachnoid hemorrhage, neurological deficits, and mortality. Results- In the milder model, VNS significantly reduced aneurysm rupture rate compared with FNS (29% versus 80%, respectively). Subarachnoid hemorrhage grades were also lower in the VNS group. In the more severe model, both VNS and FNS arms developed very high rupture rates (77% and 85%, respectively). However, VNS significantly improved the survival rate compared with FNS after rupture (median survival 13 versus 6 days, respectively), without diminishing the subarachnoid hemorrhage grades. Chronic daily VNS reduced MMP-9 (matrix metalloproteinase-9) expression compared with FNS, providing a potential mechanism of action. As an important control, chronic daily VNS did not alter systemic arterial blood pressure compared with FNS. Conclusions- VNS can reduce aneurysm rupture rates and improve the outcome from ruptured aneurysms.

Keywords: elastase; hypertension; inflammation; intracranial aneurysm; subarachnoid hemorrhage.

Figures

Figure 1.. Experimental protocols and morphological endpoints

(A) Experimental timeline shows the timing of aneurysm induction model procedures and VNS or FNS (red triangles). Mild and severe model differences in DOCA dosing and high-salt diet are also shown. Transcutaneous electrical stimulus to achieve VNS or FNS consisted of 1 msec, 5 kHz sine wave pulses delivered at 25 Hz for 2 minutes, repeated once 5 minutes apart. (B) Severe model consistently yielded higher arterial blood pressures than the mild model (p

Figure 2.. Primary outcomes

(A) Survival (upper row) and deficit-free survival (lower row) curves for FNS and VNS are shown for mild (left) and severe (right) models (n=10 FNS and 14 VNS in the mild model, and 13 each in the severe model; Gehan-Breslow-Wilcoxon test). (B) Neurological deficit grades for mild (left) and severe (right) models. Each data point reflects one animal (Mann-Whitney test). (C) Rupture rates (upper row) and SAH grades (lower row) are shown for mild (left) and severe (right) models. Rupture rates were compared between FNS and VNS using Fisher’s exact test, and SAH grades using Mann-Whitney test.

Figure 3.. Secondary outcomes

(A) Aneurysm count per animal (upper row) and aneurysm diameters (lower row) are shown for mild (left) and severe (right) models (numbers of mice: 10 FNS, 14 VNS in the mild model, and 13 each in the severe model). Aneurysm counts were compared between FNS and VNS using unpaired t-test. Aneurysm diameters (number of aneurysms: 19 FNS, 22 VNS in the mild model, 30 FNS, 28 VNS in the severe model) were compared between FNS and VNS using Mann-Whitney test for non-normally distributed data. (B) Aneurysm size and distribution maps for mild (left) and severe (right) models are shown for FNS (black) and VNS (red). Blue “+” indicates injection site. (C) Aneurysm distance to the injection site for mild (left) and severe (right) models are shown (number of aneurysms as in B; unpaired t-test with Welch’s correction for unequal variances).

Figure 4.. Pro-inflammatory gene expression within the circle of Willis.

Exploratory data are shown on mRNA expression of pro-inflammatory genes within the circle of Willis in FNS- or VNS-treated mice 4 days after elastase injection (n=5 FNS, 6 VNS; unpaired t-test).