Modulation of neuroinflammation and memory dysfunction using percutaneous vagus nerve stimulation in mice

Abstract

Background: The vagus nerve is involved in regulating immunity and resolving inflammation. Current strategies aimed at modulating neuroinflammation and cognitive decline, in many cases, are limited and ineffective.

Objective: We sought to develop a minimally invasive, targeted, vagus nerve stimulation approach (pVNS), and we tested its efficacy with respect to microglial activation and amelioration of cognitive dysfunction following lipopolysaccharide (LPS) endotoxemia in mice.

Methods: We stimulated the cervical vagus nerve in mice using an ultrasound-guided needle electrode under sevoflurane anesthesia. The concentric bipolar needle electrode was percutaneously placed adjacent to the carotid sheath and stimulation was verified in real-time using bradycardia as a biomarker. Activation of vagal fibers was confirmed with immunostaining in relevant brainstem structures, including the dorsal motor nucleus and nucleus tractus solitarius. Efficacy of pVNS was evaluated following administration of LPS and analyses of changes in inflammation and behavior.

Results: pVNS enabled stimulation of the vagus nerve as demonstrated by changes in bradycardia and histological evaluation of c-Fos and choline acetyltransferase expression in brainstem nuclei. Following LPS administration, pVNS significantly reduced plasma levels of tumor necrosis factor-α at 3 h post-injection. pVNS prevented LPS-induced hippocampal microglial activation as analyzed by changes in Iba-1 immunoreactivity, including cell body enlargement and shortened ramifications. Cognitive dysfunction following endotoxemia was also restored by pVNS.

Conclusion: Targeted cervical VNS using this novel percutaneous approach reduced LPS-induced systemic and brain inflammation and significantly improved cognitive responses. These results provide a novel therapeutic approach using bioelectronic medicine to modulate neuro-immune interactions that affect cognition.

Keywords: Cognition; Cytokines; Microglia; Neuroinflammation; Percutaneous; Vagus nerve stimulation.

Conflict of interest statement

Conflicts of interest

None.

Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Figure 1:

Schematic representation of percutaneous method. A)Illustration of method. The animal is in supine position with the ultrasound transducer placed over the shaved cervical region. Anatomical landmarks used in needle positioning include: (a) ventral aspect of cervical region, (b) neck muscles, and (c) carotid artery with blood flow confirmed with Doppler imaging. Needle electrode (d) is visualized and the tip is positioned at the carotid sheath of the vagus for effective nerve stimulation (for a detailed view see Suppl. Video).B) Representative example of VNS-induced bradycardia. The period of stimulation is denoted with the black bar. Heart rate (HR) is markedly decreased with stimulation (time = 0 sec) and quickly recovers (time = 15 sec). Bradycardia is defined as a 10% reduction in HR and is plotted as the dashed line (BCT). Stimulation is applied as biphasic pulses at 20 Hz.

Figure 2:

c-Fos and ChAT activation in brainstem nuclei after pVNS.A) Schematic image from Allen Mouse Brain Atlas [http://mouse.brain-map.org/] illustrating the NTS and DMX region followed by representative images of c-Fos staining in naïve, sham, and 30 min pVNS mice. Images taken of brainstem region ipsilateral to stimulation after 20 Hz stimulation. Scale bar: 50 μm.B) Quantification of c-Fos+ cells in the ipsilateral and contralateral NTS 1 h after pVNS. pVNS significantly induced c-Fos expression in the ipsilateral NTS. C) Quantification of c-Fos+ cells in the ipsilateral and contralateral DMX 1 h after pVNS. Bilateral c-Fos+ activation was detected after pVNS in the DMX.D) Representative images of ChAT (left) and c-Fos (middle) in the DMX (scale bar: 50 μm). Double-labeled neurons are visible in yellow (right) in the merged image. E) Quantification of ChAT+/c-Fos+ double-labeled cells following pVNS. Bilateral activation was evident in the DMX following 30 min pVNS. Abbreviations: nucleus tractus solitarius (NTS), dorsal motor nucleus of the vagus (DMX), area postrema (AP), central canal (CC), vagus nerve stimulation (VNS), choline acetyltransferase (ChAT). Data are presented as means ± SEMs and analyzed by two-way ANOVA and Tukey’s post-hoctests. N= 5 mice/group for panels B & C, and N = 3 for panel E *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as indicated.

Figure 3:

Effects of pVNS on systemic TNF-α induction after endotoxemia.A) Preemptive pVNS, 10 or 20 Hz stimulation, significantly reduced LPS-induced TNF-α upregulation at 3 h (n=5–7; ***P<0.0001 and **P=0.001 respectively). B) Rescuing effects of pVNS after LPS challenge. 10 Hz pVNS, but not 20 Hz stimulation, was able to reduce plasma TNF-α levels (n=5–7; ***P<0.0001). The data are presented as means ± SEMs and analyzed byt−tests. N=5–7 mice/group.

Figure 4:

Morphological changes in microglia following pVNS and LPS in hippocampus dentate gyrus region. A) Representative images of Iba-1 immunoreactivity in the DG in naïve, LPS (1 mg/kg), 10 and 20 Hz pVNS before LPS administration and representative segmented microglia cells from each of the experimental groups indicating the key morphological features quantified after pVNS and LPS treatments. Scale bars: 50 μm (top panel), 10 μm (bottom panel).B) Mean cell counts of ramified compared to non-ramified microglia. LPS induced significant changes in microglia morphology with a shift from ramified to non-ramified cells. Both 10 or 20 Hz pVNS reduced LPS-induced non-ramified microglial morphology. The 10 Hz pVNS restored ramified microglial morphology, but the 20 Hz stimulation was not significant. C)Representative images of Iba1+/CD68+ from the DG region across experimental groups. Scale bar: 10 μm. D)Quantification of the percent area of CD68 within Iba1+ cells across experimental groups. The data are presented as means ± SEMs and were analyzed by two-way ANOVA and Tukey’s post-hoc tests and for CD68 % Area, Kruskal–Wallis, followed by Dunn’s test. N=3 mice/group, *P<0.05, ****P<0.0001, LPS ramified vs. other ramified groups; ^P<0.05, LPS non-ramified vs. other non-ramified groups.

Figure 5:

pVNS rescues LPS-induced cognitive deficits. A) Object preference scores for the “What”, “Where”, and “When” memory task for naïve, 1 mg/kg (i.p.) LPS-t, 10 Hz + LPS-, and 20 Hz + LPS-treated mice. Animals were trained and tested 24 h after treatments. #P<0.05, naïve vs other groups; ^P<0.05, LPS vs other groups. B)Object exploration times for the novel and now familiar objects in a memory load experiment that sequentially increased the numbers of total objects from 1 to 7 across the 7 consecutive trials. The same mice were used as in the previous episodic memory experiment, except they were tested at 48 h after treatments. The data are presented as means ± SEMs and analyzed by RMANOVA followed by Bonferroni corrected post-hoc tests. N=16 mice in the LPS group and 8 mice each in the other three groups. *P<0.05, novel vs. other objects in a given trial; #P<0.05, naïve vs other groups; ^P<0.05, LPS vs other groups; +P<0.05, between subjects effects vs. 10Hz+LPS.