The Role of Vagus Nerve on Dexmedetomidine Induced Survival and Lung Protection in a Sepsis Model in Rats.

Background: Sepsis often results in acute lung injury (ALI). Sedative dexmedetomidine 21 (Dex) was reported to protect cells and organs due to its direct cellular effects. This study 22 aims to investigate the role of vagus nerves on Dex induced lung protection in a model of 23 lipopolysaccharide (LPS)-induced ALI in rats. 24 Methods: The bilateral cervical vagus nerve of male Sprague-Dawley rats was sectioned or 25 just exposed without section as sham surgery. The ALI was induced by intraperitoneal 26 injection of LPS (1 or 10 mg/kg). After LPS administration, Dex antagonist yohimbine (YOH) 27 (1 mg/kg) and/or Dex ( 25 μg/kg) was injected intraperitoneally at 0, 4, 8 and 12 hours to rats 28 with or without vagotomy. The severity of ALI was determined with survival curve analysis 29 and lung pathological scores of haematoxylin and eosin (H-E) staining sections. The plasma 30 concentrations of interleukin 1beta (IL- 1β), tumour necrosis factor -alpha (TNF- α), 31 catecholamine (CA) and acetylcholine (Ach) were measured with enzyme-linked 32 immunosorbent assay (ELISA). 33 Results: The median survival time of LPS-induced ALI rats was significantly prolonged by 34 Dex (22 hours, 50% CI, [31.25, 90.63]) compared that in the LPS group (14 hours, 50% CI, 35 [18.75, 81.25], P < 0.05), and the acute lung injury score was significantly reduced by Dex 36 (6.5, 50% CI, [5.75, 7.5] vs 11.5, 50% CI, [10.75, 12.50] in the LPS group, P < 0.01). 37 However, these protective effects of Dex were significantly reduced by either YOH 38 administration or vagotomy. Dex significantly decreased LPS-induced plasma IL- 1β (pg/ml) 39 (20.75 ± 0.78 vs. 30.22 Our current study found in a sepsis model of rats that dexmedetomidine prolonged the survival time and provided lung protection. Its effects were significantly attenuated by vagotomy and/or blocking α 2 -adrenoceptor blocker, indicating that anti-inflammation and cytoprotection of Dex were likely through high vagal nerve tone and α 2 -adrenoceptor activation. Our work further highlighted that the parasympathetic nerve system is important in maintaining body’s hemostasis under physiological and pathophysiological condition, indicating that even partial vagotomy, e.g., during upper gastrointestinal surgery, should be avoided whilst, in any case, once vagotomy accidently occurred, then the favorable effects of Dex may likely be reduced.


Background 50
The autonomic nervous system plays an essential role in inflammation and immune 51 modulation (Benarroch 2019); for example, vagal nerve stimulation can suppress both local 52 and systemic inflammation (Borovikova, Ivanova, Nardi, et al. 2000). Besides, previous 53 studies ( Vida et al. 2011;Pavlov and Tracey 2015) also demonstrated that vagus nerve 54 decreased the levels of inflammatory cytokine such as tumor necrosis factor (TNF) and 55 interleukin (IL), which are the key mediator of the inflammatory response in LPS-induced 56 endotoxemia; these effects may be closely associated with its anti-inflammatory effect 57 through the cholinergic anti-inflammatory neural pathway. Indeed, stimulation applied to the 58 vagus nerves elevated the level of acetylcholine, a neurotransmitter, that interacts with α7 59 subunit of the nicotinic acetylcholine receptor (α7nAChR). It was also reported that vagus 60 nerve stimulation or pre-treatment with an α7nAChR agonist reduced neuroinflammation 61 while vagotomy or using selective a7nAChR antagonists enhanced the inflammatory response 62 (Zhu et al. 2016). inflammation and oxidative stress, and protect against inflammation and oxidative 67 stress-related diseases ). Clinically, Dex was reported to significantly reduce 68 inflammatory factors IL-1β, IL-6 and TNF-α, and intra-abdominal pressure in patients with 69 sepsis after abdominal surgery, compared to propofol (Tasdogan et al. 2009). In addition, 70 previous studies have found that the parasympathetic nerve played an important role in the 71 anti-inflammatory effect of Dex, which may activate the efferent vagus nerve and led to the 72 release of anti-inflammatory factors (Xiang et al. 2014). We, therefore, designed the current 73 study to investigate the role of vagus nerve on Dex against mortality and LPS-induced ALI in 74 rats.

Sepsis model, vagotomy and drug administration 85
Rats were anesthetized with intraperitoneal injection of sodium pentobarbital (Zizheng,86 Shanghai, China) (30 mg/kg) and then randomly subjected to vagus nerves exposure only 87 (sham surgery) in the carotid sheaths through a median skin incision or then cut in both side 88 (vagotomy group). Thirty minutes after surgery, rats were injected intraperitoneally with 1 or 89

Experimental groups 96
In the sham surgery cohorts, they were randomly subdivided into 5 groups according to 97 injections of normal saline, Dex, LPS, LPS plus Dex, or LPS plus YOH and Dex. In the 98 vagotomy cohorts, they were randomly subdivided into 4 groups according to injections of 99 normal saline, Dex, LPS, LPS plus Dex, or LPS plus YOH and Dex. All 9 groups (n = 8) were 100 subjected to survival assessment (Fig. 1). Their respiration and locomotor activities were 101 closely monitored. 102 103

Blood and lung tissue sampling and analyses 104
New cohorts of 9 groups (n = 6-10) illustrated in Fig. 1

Statistical analysis 126
The survivals and ALI scores were expressed as median and 50% confidence interval (median, 127 50% CI [25 th percentiles, 75 th percentiles]), which was presented as survival curve and box 128 plot, respectively. The data of ELISA was expressed as mean ± SD together with dots plot. 129 Statistical analysis was performed with GraphPad Prism 7.0 software (GraphPad Software, La 130 Jolla, California, USA). Survival data were analyzed using a Kaplan-Meier curve and a 131 log-rank test. If normally distributed, the data was analyzed with one-way analysis of variance 132 (ANOVA) followed by Tukey's multiple comparisons test, or otherwise, the Dunnet T3 test 133 was used. A p-value < 0.05 was considered as a statistical significance.

Vagus nerve integrity is involved in Dex protective effect 137
The mortality rate of the vagotomy (VAG) group was 100% at the 80 hours of the 138 experimental period and the median survival time was 14 hours (50% CI, [12.50, 75.00]); 139 Dex significantly increased survival to 25% and the median survival time was 24 hours (50% 140 CI, [12.50, 90.63]) (p = 0.008) ( Fig. 2A). To determine the dose effects of LPS on survival, 1 141 mg/kg and 10 mg/kg LPS were used in rats without vagotomy. The mortality induced by the 142 low dose of LPS with or without Dex and YOH was the same to be 16.7% and there was no 143 difference of survival between two groups (Fig 2B) (Fig. 2C). Yohimbine abolished the effects of Dex (Fig. 2C) induced by the low dose of LPS after vagotomy (Fig. 2D) the high dose of LPS after vagotomy (Fig. 2E). Yohimbine abolished these effects of Dex 151 (Fig. 2D, E). superposed with LPS and/or Dex had no further effects on these measurements (Fig. 4).

Availability of data and materials 319
The datasets used and/or analysed during the current study are available from the 320 corresponding author on reasonable request. 321 322

Competing interests 323
The authors declare that they have no competing interests.   Experimental grouping and sub-cohorts' assignment. Rats were anesthetized with sodium pentobarbital for vagotomy or sham surgery (only exposure of vagus nerves without vagotomy) followed with various treatments for survival and lung pathological assessment and blood biomarker measurements. VAG, vagotomy; LPS, lipopolysaccharide; Dex, dexmedetomidine; YOH, yohimbine.

Figure 4
The anti-in ammatory effect of Dex may be related with the integrity of the vagus nerve. Serum samples were collected 16 hours after LPS administration in the absence or presence of Dex or YOH or VAG. The levels of IL-1β (A) and TNF-α (B) in serum of different groups were evaluated with ELISA analysis. The data was expressed as mean ± SD and dots plot (n = 6-10). *p < 0.05, **p< 0.01 versus the CON group; #p< 0.05, ##p< 0.01 versus the VAG + LPS (10) + Dex group. IL-1β, interleukin 1-beta; TNF-α, tumor necrosis factor alpha; CON, control; VAG, vagotomy; LPS, lipopolysaccharide; Dex, dexmedetomidine; YOH, yohimbine.