Vagus nerve stimulation protects against cerebral injury after cardiopulmonary resuscitation by inhibiting inflammation through the TLR4/NF-κB and α7nAChR/JAK2 signaling pathways.

BACKGROUND: Our previous research proved that vagus nerve stimulation (VNS) improved the neurological outcome after cardiopulmonary resuscitation (CPR) by activating α7 nicotinic acetylcholine receptor (α7nAChR) in a rat model, but the underlying mechanism of VNS in neuroprotection after CPR remains unclear. METHODS: In vivo, we established a mouse model of cardiac arrest (CA)/CPR to observe the survival rate, and the changes in inflammatory factors and brain tissue after VNS treatment. In vitro, we examined the effects of α7nAChR agonist on ischemia/reperfusion (I/R)-induced inflammation in BV2 cells under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions. We observed the changes in cell survival rate, the levels of inflammatory factors, and the expressions of α7nAChR/Janus kinase 2 (JAK2) and toll-like receptor 4 (TLR4) /nuclear factor-κB (NF-κB). RESULTS: In vivo, VNS preconditioning enhanced functional recovery, improved the survival rate, and reduced hippocampal CA1 cell damage, and the levels of inflammatory mediators after CA/CPR. The application of α7nAChR agonists provided similar effects against cerebral injury after the return of spontaneous circulation (ROSC), while α7nAChR antagonists reversed these neuroprotective impacts. The in vitro results mostly matched the findings in vivo. OGD/R increased the expression of tumor necrosis factor-alpha (TNF-α), TLR4 and NF-κB p65. When nicotine was added to the OGD/R model, the expression of TLR4, NF-κB p65, and TNF-α decreased, while the phosphorylation of JAK2 increased, which was prevented by preconditioning with α7nAChR or JAK2 antagonists. CONCLUSION: The neuroprotective effect of VNS correlated with the activation of α7nAChR. VNS may alleviate cerebral IR injury by inhibiting TLR4/NF-κB and activating the α7nAChR/JAK2 signaling pathway.

[Preliminary study on the efficacy of ultrasound therapy in the rat model of sepsis].

OBJECTIVE: To investigate the possible mechanism of ultrasound therapy in the rat model of sepsis. METHODS: Seventy-eight male Sprague-Dawley (SD) rats were randomly divided into Sham group (n = 12), septic model group (n = 22), ultrasound treatment group (n = 22), methyllycaconitine citrate (MLA) combined with ultrasound treatment group (n = 22). In the Sham group, only the abdomen was opened, the cecum was found to be free, without cecal ligation and puncture (CLP). In the septic model group, CLP was used to replicate the septic rat model. After operation, each group of rats were subcutaneously injected with preheated 37 centigrade normal saline. The rats in the ultrasound treatment group were treated with ultrasound [Philips IU22 L9-3 ultrasound instrument and 9 MHz probe were used to break the sequence in the spleen area once every 6 seconds, with 1 second for each time, the mechanical index (MI) was 0.72, and the treatment time was 10 minutes]. In the MLA combined with ultrasound treatment group, α7 nicotinic acetylcholine receptor (α7nAChR) specific blocker MLA 4 mg/kg was injected intraperitoneally 30 minutes before operation, and ultrasound treatment was performed 2 hours after operation. The levels of tumor necrosis factor-α (TNF-α) and interleukin (IL-1ß, IL-6) in serum of each group were measured by enzyme-linked immunosorbent assay (ELISA) at 24 hours after operation. The 10-day survival rate of each group was recorded, and the symptoms of each group were evaluated by clinical disease score (CDS). The histopathological changes of lung and colon were observed under light microscope. RESULTS: Compared with the Sham group, the 10-day survival rate of rats in the septic model group was decreased significantly [40% (4/10) vs. 100% (6/6)], the CDS was (10.73±2.19 vs. 6.17±0.58) and the levels of TNF-α, IL-6, and IL-1ß were increased significantly at 24 hours after operation [TNF-α (ng/L): 42.00±8.92 vs. 13.16±3.19, IL-6 (ng/L): 129.37±25.04 vs. 63.99±12.92, IL-1ß (ng/L): 254.98±67.27 vs. 76.83±25.39, all P < 0.01]. Compared with the septic model group, the survival rate in the ultrasound treatment group was improved [70% (7/10) vs. 40% (4/10)], but there was no significant difference (P > 0.05). The CDS (7.64±2.68 vs. 10.73±2.19) and the expressions of TNF-α, IL-6, and IL-1ß were significantly reduced at 24 hours after operation [TNF-α (ng/L): 16.93±6.02 vs. 42.00±8.92, IL-6 (ng/L): 73.65±24.38 vs. 129.37±25.04, IL-1ß (ng/L): 111.86±14.08 vs. 254.98±67.27, all P < 0.01]. Compared with the ultrasound treatment group, the survival rate in the MLA combined with ultrasound treatment group was reduced [60% (6/10) vs. 70% (7/10)], but the difference was not statistically significant (P > 0.05). CDS was significantly increased (9.55±2.72 vs. 7.64±2.68), and the levels of TNF-α, IL-6 and IL-1ß were significantly increased at 24 hours after operation [TNF-α (ng/L): 34.61±7.89 vs. 16.93±6.02, IL-6 (ng/L): 112.92±10.42 vs. 73.65±24.38, IL-1ß (ng/L): 212.57±32.16 vs. 111.86±14.08, all P < 0.01]. Microscopically, in the septic model group, the alveolar septum was thickened, a large number of inflammatory cells infiltrated, normal pulmonary reticular structure disappeared, and pulmonary interstitium showed obvious hemorrhage and edema, meanwhile, the structure of colonic villi was obviously abnormal, with cells were edema and inflammatory cell infiltration, and the arrangement was disordered, so that the subepithelial space and the top of it fell off. After ultrasound treatment, the thickness of the alveolar interval in rats was similar to that in Sham group, without obvious inflammatory cell infiltration, and the pulmonary reticular structure was relatively intact. At the same time, the morphology of colonic villi was basically normal and orderly, the edema of cell was not obvious, and subcutaneous space and tip fall off were not obvious. After being antagonized by MLA, the rat lung tissue showed thickened alveolar septum, inflammatory cell infiltration, incomplete pulmonary network structure, hemorrhage and edema in the interstitium. The villi structure of the colon was faintly visible, with obvious cell edema and inflammatory cell infiltration, and the arrangement was abnormal. CONCLUSIONS: Ultrasound treatment improves the prognosis of septic rats, MLA can reverse the anti-inflammatory effect of ultrasound therapy by antagonizing α7nAChR, suggesting that the protective mechanism of ultrasound in sepsis may be related to activating the cholinergic anti-inflammatory pathway mediated by α7nAChR.

Left-sided vagus nerve stimulation improves cardiopulmonary resuscitation outcomes in rats as effectively as right-sided vagus nerve stimulation.

BACKGROUND: Our group previously reported that right-sided vagus nerve stimulation (RVNS) significantly improved outcomes after cardiopulmonary resuscitation (CPR) in a rat model of cardiac arrest (CA). However, whether left-sided vagus nerve stimulation (LVNS) could achieve the same effect as RVNS in CPR outcomes remains unknown. METHODS: A rat model of CA was established using modified percutaneous epicardial electrical stimulation to induce ventricular fibrillation (VF). Rats were treated with LVNS or RVNS for 30 minutes before the induction of VF. All animals were observed closely within 72 hours after return of spontaneous circulation (ROSC), and their health and behavior were evaluated every 24 hours. RESULTS: Compared with those in the RVNS group, the hemodynamic measurements in the LVNS group decreased more notably. Vagus nerve stimulation (VNS) decreased the serum levels of tumor necrosis factor-alpha (TNF-α) and the arrhythmia score, and attenuated inflammatory infiltration in myocardial tissue after ROSC, regardless of the side of stimulation, compared with findings in the CPR group. Both LVNS and RVNS ameliorated myocardial function and increased the expression of α-7 nicotinic acetylcholine receptor in the myocardium after ROSC. Moreover, a clear improvement in 72-hour survival was shown with VNS pre-treatment, with no significant difference in efficacy when comparing the laterality of stimulation. CONCLUSIONS: LVNS may have similar effects as RVNS on improving outcomes after CPR.

Ultrasound improves the outcomes of cardiopulmonary resuscitation in rats by stimulating the cholinergic anti­inflammatory pathway.

The present study investigated the effects of the ultrasound (US), a noninvasive technique, on ischemia­reperfusion injury (IRI) following cardiopulmonary resuscitation (CPR). The animals used in the present study were randomized into five groups (n=8 per group) as follows: i) The CPR group, where the rats underwent 6 min of untreated ventricular fibrillation (VF) followed by CPR and defibrillation; ii) the US group, in which the treatment was identical to the CPR group with the exception that rats were exposed to US treatment 24 h prior to CPR; iii) the MLA group, in which the treatment was identical to the US group with the exception that the α7 nicotinic acetylcholine receptor (α7nAChR) antagonist MLA (4 mg/kg) was administered 30 min prior to US and VF respectively; iv) the GTS group, in which the treatment was identical to the CPR group with the exception that the α7nAChR agonist GTS­21 (4 mg/kg) was injected 30 min prior to VF; and v) the SHAM group, in which the rats were exposed to surgical preparation without CPR and US application. At 1 day prior to CPR, the US treatment was administered to the left kidney by US pulses (contrast general mode with 9 MHz) with a bursting mechanical index of 0.72 for 2 min. Following treatment of the left kidney, the right kidney was exposed to identical US treatment for an additional 2 min. The results demonstrated that US preconditioning decreased the number of defibrillations required and shortened the duration of CPR. US also suppressed tumor necrosis factor­α and interleukin­6 levels following resuscitation (P<0.05), and a significantly longer overall survival time was observed in the US­treated animals (P<0.01). In addition, US attenuated neuronal injury and promoted the expression of α7nAChR in hippocampal neurons (P<0.05). However, the protective effects of US were abolished by MLA and imitated by GTS­21. The results of the present study demonstrated that prior exposure to US may improve animal outcomes following CPR, and the protective effects of US may be dependent on the cholinergic anti­inflammatory pathway (CAP) via α7nAChR.

Xuezhikang improves the outcomes of cardiopulmonary resuscitation in rats by suppressing the inflammation response through TLR4/NF-κB pathway.

BACKGROUND/AIMS: Xuezhikang (XZK), a red yeast rice extract with lipid-lowering effect, contains a family of naturally statins, such as lovastatin. In recent years, its effect beyond the regulation of lipids has also been received increasing attention. Therefore, the purpose of this study was to explore the protective effects and possible molecular mechanisms of XZK on brain injury after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR), and to investigate whether it has a dose-dependent effect and the difference with lovastatin. METHODS: Rats were treated with low-dose XZK (XZK-L, 20 mg/kg/d), high-dose XZK (XZK-H, 200 mg/kg/d) and lovastatin by gavage once daily for 2 weeks before CA. The levels of TNF-α, IL-6 and IL-1ß were evaluated at 1, 4, and 72 h post-CA/CPR. The survival rate, neurological deficit score (NDS), and expression of TLR4, phosphorylated NF-κB and TNF-α in hippocampal tissues were evaluated at 72 h post-CA/CPR. RESULTS: CA/CPR induced a significant increase in serum TNF-α, IL-6 and IL-1ß, as well as increased expressions of TLR4, phosphorylated NF-κB and TNF-α in the hippocampus. Both low-dose and high-dose XZK treatment inhibited the expression of these inflammatory cytokines. In addition, it reduced the number of defibrillations and shortened the duration of CPR required for return of spontaneous circulation (ROSC). XZK treatment also improved neurological function and 72-hour survival rate in rats. However, high-dose XZK was superior to lovastatin in the suppression of IL-1ß mRNA level and TNF-α protein level in hippocampal tissue after CPR. There were no significant differences observed among high-dose XZK, low-dose XZK and lovastatin groups in other respects. CONCLUSION: These results indicated that XZK had a protective effect against brain injury post-CA/CPR. The mechanisms underlying the protective effects of XZK may be related to the suppressing of CA/CPR-induced inflammatory response through the inhibiting TLR4/NF-κB signaling pathway.