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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 ℃ 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-1β, 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-1β 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-1β(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-1β 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-1β(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-1β 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-1β(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. Conclusion: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.
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Objective:To explore the protective effect of vagus nerve stimulation (VNS) on the prognosis of rats suffering from cardiac arrest/cardiopulmonary resuscitation (CA/CPR) under different treatment timings.Methods:The method of percutaneous epicardial electrical stimulation was used to establish CA model of rat. Fifty-three male SD rats were randomly (random number) divided into the sham group ( n=5), CPR group ( n=12), PRE group ( n=12), POST5 group ( n=12) and POST30 group ( n=12). The sham group did not experience CA/CPR. VNS treatment was started at 30 min before CA (PRE group, n=12), 5 min after recovery of spontaneous circulation (ROSC) (POST5 group, n=12), and 30 min after ROSC (POST30 group, n=12) in different VNS-treated group, respectively. The electrical stimulation was applied to the vagus nerve for 30 min with a unified parameter. The neurological deficit scores at 24, 48, and 72 h after ROSC were recorded, and the survival rate in each group was observed. TUNEL staining was used to detect the apoptosis of cortical area and the expression of α7 nicotinic acetylcholine receptor (α7nAChR) in brain tissue was measured by immunofluorescence at 72 h after ROSC. Variables were compared with one-way analysis of variance, and survival for Kaplan-Meier curves were tested with the log-rank test. A P value less than 0.05 was considered statistically significant. Results:Compared with the CPR group (survival rate 33.33%), both pre-treatment (survival rate 75%) and post-treatment of VNS (POST5 group survival rate 75% and POST30 group survival rate 83.33%) significantly improved the 72 h survival rate after CPR ( P<0.05), mitigated neurological deficits after ROSC, reduced the positive rate of apoptosis neurons, and up-regulated the expression of α7nAChR in cerebral cortex. There was no significant difference among the VNS-treated groups (all P>0.05). Conclusions:Both pre-treatment and post-treatment of VNS can play a protective role in rats after CA/CPR, which may be related to the activation of α7nAChR-mediated anti-inflammatory and anti-apoptosis effects.
ABSTRACT
Objective To establish the cardiac arrest (CA) model in rats by modified transcutaneous electrical stimulation on epicardium. Methods This study was performed in the Emergency Medicine laboratory in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. After 10 Sprague-Dawley male rats weighing 330-380 g were anesthetized, two acupuncture needles connected to the anode and cathode of a stimulator were transcutaneously inserted into the epicardium as electrodes. The puncture points were located quantitatively according to the anatomical structure of the rat chest. The electrical stimulation was maintained for 3 minutes to induce ventricular fibrillation(VF). Cardiopulmonary resuscitation (CPR) included chest compressions, intravenous adrenaline and defi brillation operated at 6 min after a period of nonintervention. Results CA was induced after the implement of the effective electrical stimulation in all ten rats in this experiment. The average current intensity to induce VF was (1.80 ± 0.59) mA, the average time to induce CA was (5.07 ± 2.37)s,the average time of the total electrical stimulation was(187.50 ± 12.75)s and the total time of CA was 6 min. At the end of the electrical stimulation, 9 rats presented VF and 1 rat showed pulseless electrical activity. The restoration of spontaneous circulation was achieved in all 10 rats. The average time of CPR was(190.90±68.60) s, the mean numbers of defi brillation were(1.20 ± 0.63) , and he average number of adrenaline application were (1.20 ± 0.42) times. Neither visible hemorrhage on epicardium nor gross pulmonary congestion was observed. Conclusions The modified transcutaneous electrical stimulation on epicardium to produce CA model in rats is an easily applicable and effective technique. This model may provide an alternative for experimental research of CPR.