Vagus nerve stimulation ameliorates cognitive impairment caused by hypoxia

Abstract

Introduction: Hypoxia significantly impairs cognitive function due to the brain's high demand for oxygen. While emerging evidence suggests that vagus nerve stimulation (VNS) can enhance cognition, its effectiveness in mitigating behavioral and molecular impairments caused by hypoxia remains unknown. This study investigated whether VNS could alleviate hypoxia-induced deficits in cognitive performance and neurotrophin expression in rats.

Methods: Healthy male Sprague-Dawley rats were randomly assigned to three groups: sham, hypoxia, and VNS + hypoxia. VNS was delivered during hypoxia (8% oxygen) exposure using 100 μs biphasic pulses at 30 Hz and 0.8 mA. Cognition and performance were assessed by behavioral testing and hippocampal tissue was collected for molecular analysis. NGF and BDNF mRNA levels were measured by quantitative PCR, and protein expression was evaluated by immunohistochemistry.

Results: The passive avoidance test (PAT) performance was significantly reduced by hypoxia exposure compared to the sham group, and administration of VNS during hypoxia ameliorated this impairment. Hypoxia significantly reduced NGF and BDNF mRNA levels in the hippocampus 24 h post-exposure. VNS restored NGF mRNA to sham levels and partially increased BDNF mRNA. Immunohistochemistry results showed VNS significantly restored NGF protein expression in the hippocampus, while BDNF levels remained unchanged.

Discussion: These findings suggest that VNS may serve as a promising intervention for cognitive impairments induced by hypoxia.

Keywords: BDNF; NGF; hypoxia; learning; memory; rats; vagus nerve stimulation.

Figure 1

Experimental design. After recovery from surgical VN cuff implantation, all rats were utilized for behavioral, RT-qPCR and IHC analysis. Animals were habituated to the behavior and VNS/Hypoxia arenas on habituation day. The training day, 24 h after the habituation day, involved alternating periods of hypoxia exposure and VNS for a total of four hours of hypoxia and two hours of VNS, interspersed with behavior training and EZM testing. About 24 h after the training day, the rats were tested in the NOR and PAT paradigms. All animals were euthanized within two hours after completing behavior testing. Brain tissues were separated for left and right cerebral hemispheres and were either post-fixed in 4% PFA for IHC or dissected into different brain regions and stored in RNA-later for RT-qPCR.

Figure 2

VNS mitigates hypoxia induced cognitive impairment in male rats. (A) The time spent in the open arm was not different for male rats in the sham, hypoxia, or VNS + hypoxia groups. (B) Hypoxia significantly decreased the latency to cross into the dark chamber compared to the sham rats by post-hoc analysis. VNS significantly increased the time to cross as compared to rats exposed to hypoxia based on post-hoc analysis. (C) In the NOR test, a two-way ANOVA found a significant effect of “object” for object exploration time, with post-hoc analysis showing this effect to be significant within the SHAM group. (D) A two-way ANOVA found a significant effect of ‘Object’ for frequency of object exploration, though post-hoc analysis did not find this effect to be significant within the groups. (E) Novel Object Preference was not significantly different among the groups. Data are presented as the mean ± SEM; *p < 0.05, **p < 0.01.

Figure 3

Hypoxia Decreases NGF and BDNF mRNA Expression in Hippocampus; VNS Restores Only NGF mRNA expression. (A) Post-hoc analysis identified NGF mRNA expression was significantly reduced between the sham and hypoxia groups. A significant increase in NGF mRNA between the hypoxia and VNS + hypoxia group was also identified by post-hoc analysis. (B) BDNF mRNA expression was significantly decreased with hypoxia exposure based on post-hoc analysis. Each bar graph represents the mean ± SEM; *p < 0.05 **p < 0.01.

Figure 4

VNS mitigates hypoxia-induced reduction in NGF protein expression in the hippocampus. (A) Hypoxia and VNS augmented NGF protein expression in different regions of the hippocampus. (B) In the CA1 region, VNS significantly increased NGF protein expression compared to the hypoxia group as identified by post-hoc analysis. (C) In the CA2 region, no significant changes in NGF protein expression were detected between groups by one-way ANOVA. (D) Post-hoc analysis identified a significant reduction in NGF protein levels during hypoxia exposure when compared to the sham or VNS + hypoxia groups in the CA3 region. (E) Post-hoc analysis identified a significant reduction in NGF protein expression during hypoxia exposure when compared to the sham or VNS + hypoxia groups in the DG. Each bar graph represents the mean ± SEM; *p < 0.05, **p < 0.01. Scale bar = 50 μm.