The Effect of Subdiaphragmatic Vagotomy on Heart Rate Variability and Lung Inflammation in Rats with Severe Hemorrhagic Shock

Abstract

Background

The role of the sub-diaphragmatic branch of the vagus nerve in mediating heart rate variability (HRV) and inflammatory reaction to long term hemorrhagic shock has not been determined prior to this study.

Methods

Male Sprague-Dawley rats were divided into four groups of Sham, sub-diaphragmatic vagotomized (Vag), long term (130±2 minutes) hemorrhagic shock (LHS), and sub-diaphragmatic vagotomized with LHS (Vag+LHS). Hemodynamic parameters were recorded and HRV calculated during multiple phases of hemorrhagic shock. The expressions of TNF-α and iNOS were measured in the spleen and lung tissues at the conclusion of the protocol.

Results

Decreases in blood pressure during blood withdrawal were identical in the LHS and Vag+LHS groups. However, heart rate only decreased in the Nadir-1 phase of the LHS group. HRV indicated increased power in the very-low, low, and high (VLF, LF, and HF) frequency bands during the Nadir-1 phase of the LHS group and decreased power in the Vag+LHS group. There was metabolic acidosis partially compensated with respiratory system in the LHS and Vag+LHS groups. Increases of TNF-α and iNOS expression in the spleen and lung of the LHS group were reversed in the Vag+LHS group.

Conclusion

This study indicates that sub-diapragmatic vagotomy increases lung inflammatory reactions and blunts the cardiac vagal tone surge in response to severe hemorrhagic shock.

Background

Hemorrhagic shock is one of the common causes of death in the world (Gonzalez et al., 2007). Hemorrhagic shock is categorized into mild, moderate, and severe, depending on the different degrees of hemorrhage (Bonanno, 2012; Evans et al., 2001; Troy et al., 2003; Schadt and Ludbrook, 1991; Shenkar et al., 1994). Hemorrhagic shock is reversible in the early stages and a delay in diagnosis and/or treatment initiation can lead to extensive body organ ischemia (Shenkar et al., 1994). In this condition fluid replacement therapy may not be useful, and in fact may lead to systemic inflammatory reactions, and serious organ damage (Rushing and Britt, 2008; Niu et al., 2007). This is why early diagnosis of the presence and stage of shock is so important to prevent a catastrophic outcome (Shah et al., 1998; Paul, M., 2018). Since immediately upon the initiation of hemorrhage, hormonal and neural compensatory mechanisms are activated (Orlinsky et al., 2001; Grässler et al., 1990), the question remains whether or not the evaluation of neural activity could be beneficial in assessing the level, severity, and outcome of patients with hemorrhagic shock. The activity of the autonomic nervous system can be evaluated by heart rate variability analysis (HRV) (McCraty and Shaffer, 2015). Inter-beat interval can be extracted from electrocardiogram or pulsatile blood pressure recording as time interval between consecutive heartbeats (Aires et al, 2017). A few previous studies have shown that HRV can estimate the autonomic nervous system activity in hemorrhagic shock (Nogami et al., 2010). Furthermore, HRV has been investigated in three phases of class II hemorrhagic shock in a recent study (Khodadadi et al., 2020). Nevertheless, HRV and altered autonomic nerve activity has not been fully elucidated in different phases of severe hemorrhagic shock.

The vagus nerve is the chief parasympathetic branch of the autonomic nervous system which may play a role in the inflammatory reactions through a cholinergic anti-inflammatory reflex arc (Tonhajzerova, 2013). Spleen has been suggested to be involved in the noted reflex (Herath et al., 2019). However, the spleen is innervated by the sympathetic rather than the parasympathetic nervous system. It has been established that the sympathetic nerve fibers of the spleen are stimulated by the vagus nerve (Berthoud and Powley, 1993). Then, norepinephrine released from the sympathetic nerve fibers triggers release of acetylcholine (ACh) by resident T cells in the spleen. Consequently, ACh inhibits cytokines’ productions by the macrophages through α7 nicotinic receptors (Tracey 2007; Vida et al., 2011). However, little attention has been devoted to the role of the vagus nerve in inflammatory reactions induced by hemorrhagic shock thus far. Also, the role of the spleen in the above conditions has not been fully clarified.

One of the most vulnerable organ during hemorrhagic shock is the lung. This organ is adversely affected by inflammatory mediators released from damaged tissue (Pfeifer et al., 2013). Animal studies have shown increases of tumor necrosis factor-α (TNF-α), nuclear factor-kappa β (NF-κβ), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and malondialdehyde (MDA) in the lung tissue during hemorrhagic shock (Tsung et al., 2017). Also, the high prevalence of lung injury has been reported in patients with hemorrhagic shock (Ciesla et al., 2005). However, although the relationship between HRV, systemic hemodynamics, metabolic status, and inflammation have been expressed in different pathological conditions (Porter et al., 2009; Millis et al., 2011; Cooper et al., 2015), little has been addressed about the noted relations with or without investigating the role of the vagus nerve in hemorrhagic shock.

Based on the above background, the aim of this study was to investigate HRV, hemodynamic alterations, metabolic status, and the expression of inflammatory cytokines in the spleen and lung in long term hemorrhagic shock. Furthermore, it has been observed that manipulation of the sub-diaphragmatic vagus nerve has less off-target effect and efficiently diminishes local inflammation as compared to the cervical vagus nerve (Payne et al., 2019). So, in this study the role of left sub-diaphragmatic vagus nerve, which is closer to spleen, has been evaluated. This study was performed in conscious rats in order to extrapolate the results to trauma patients with hemorrhagic shock without the interfering effects of anesthetic drugs on the autonomic nervous system activity, respiratory system, or HRV analysis.

Methods

Study design

All experimental procedures were approved by the Center for Comparative and Experimental

Medicine and the Ethical Committee of Animal Care of Shiraz University of Medical Sciences, Shiraz, Iran, according to the provisions of the Declaration of Helsinki (No: IR.SUMS.MED.REC.1396.S203). Animals (male Sprague-Dawley rats weighing 250-300g) were housed with 12 hours light/dark cycle in controlled temperature (22 ± 2°C) and humidity of 40-50% before starting the experiments. They had free access to water and standard food. A total of 25 rats were divided into four groups of Sham (n=5), subdiaphragmatic vagotomized (Vag, n=5), long term hemorrhagic shock (LHS, n=7), and sub-diaphragmatic vagotomized with LHS (Vag+LHS, n=8). Based on a survey of similar studies (Antonino et al., 2017; Capalonga et al., 2021) we conducted a post hoc estimate of our power to detect a robust effect (d>=1.43). Given the N in all groups, we estimate we have 0.95 power to detect effects of this size and we maintain power of 0.8 to detect effects as small as d=0.83.Animals were anesthetized by intraperitoneal injection of 50 mg/kg sodium pentobarbital (Sigma, Germany). The catheters were inserted into the femoral vein (120-PE) and tail artery (50 PE), and fixed firmly. In the vagotomized groups, the left sub-diaphragmatic vagus nerves were dissected. In the Sham group, animals underwent the arterial and venous cannulations and abdominal laparotomy, whereas, the vagus nerve remained intact. The areas of surgeries were then rinsed by 1% lidocaine (Sigma, Germany) to minimize the postoperative pain in all animals. The conscious animals were transferred to an optimized dark metabolic cage (MR Plexi), and their tails were fixed outside the cage so that animals could relatively move in a cage without interrupting the hemodynamic recording. The arterial catheter was connected through a pressure transducer (MLT844) to a data acquisition system (Powerlab, PL26T04, AD instruments, Australia). The arterial blood pressure (AP) was recorded throughout the experiment at 1 kHz. After data acquisition, the AP data were downsampled to 500 Hz. The catheter of the femoral vein was used for blood sampling and blood withdrawal during induction of hemorrhagic shock (Khodadadi et al., 2020).

Sub-diaphragmatic vagotomy  

After anesthesia and cannulations of tail artery and femoral vein, an incision was made in the abdominal skin of vagotomized groups. The fascia and muscles were dissected, and the left subdiaphragmatic vagus nerve separated from the surrounding tissues and dissected. Then, muscles and skin were sutured and the surgical areas were rinsed with 1% lidocaine (Khodadadi et al., 2020; Smith et al., 1985).  

Study protocol: induction of hemorrhagic shock 

Seventy minutes after the surgeries, 100 µl of the arterial blood samples were taken for blood gas analysis. Then, the LHS and Vag+LHS groups were subjected to hemorrhagic shock by blood withdrawal through the femoral vein according to the protocols of previous studies and our pilot experiments (Khodadadi et al., 2020; Shah et al., 1998). Briefly, blood withdrawal was performed with a flow rate of 0.5 ml/min until reaching the mean arterial blood pressure (MAP) to 35±2 mm Hg. Unlike the previous study, where blood withdrawal was stopped and MAP was allowed to return toward the basal values (Khodadadi et al., 2020), in this study the blood withdrawal was continued with the flow rate of 0.25 ml/min to maintain the MAP at the noted level. This phase was called Nadir-1 and lasted about 25 minutes. Meanwhile, the heparinized collected blood was kept in ice and filtered before returning to the animals. Once the compensatory mechanisms of animals failed to keep the MAP in the range of 35±2 mm Hg, blood withdrawal was stopped and very low volumes of the heparinized and filtered blood injected constantly to maintain the MAP in the hemorrhagic shock level. An animal’s Total blood volume was estimated by use of the following formula: total blood volume (ml) = 0.06 x body weight (g) + 0.77 (Lee and Blaufox, 1985). Then, the percentage of blood loss was calculated afterward. This phase was called Nadir-2, in which 50% of the withdrawn blood was returned to the animals, and it lasted about 105 minutes. Next, the resuscitation was performed by the transfusions of the remainder of the blood (50% of collected blood) with the same amount of Lactated Ringer’s solution within 10 minutes which followed by 20 minutes of recording during the recovery phase. After that, the arterial blood samples were taken for blood gas analysis, and 1 ml of the venous blood sample taken to evaluate the plasma lactate level. Finally, animals were anesthetized with high doses of pentobarbital, and their lungs removed for molecular analysis. In the Sham and Vag groups, the time course of experiments were as long as the hemorrhagic groups. Nevertheless, these groups were not subjected to hemorrhagic shock (Fig. 1). Figure 2 indicates real traces of blood pressure and heart rate recorded by the Power Lab system in four animals in each of the experimental groups. In this figure, six time periods including steady state, Initial compensatory or In-Com (the interval that blood pressure does not change despite blood withdrawal), De-compensatory or De-Com (the period of time that blood pressure drops suddenly following a critical blood loss), Nadir 1, Nadir 2 and recovery were shown.

HRV analysis 

Three frequency bands of HRV were considered in this study. Kubios HRV premium/animal software (ver. 3.2) was used for HRV analysis. The pulse intervals of systolic blood pressures were exported from the Power lab to Kubios software, where it was analyzed following the latest guidelines for HRV analysis (Task Force 1996).  Prior to spectrum estimation, the pulse interval data were interpolated using 10 Hz cubic spline interpolation. Welch's periodogram with the Hanning window (window width of 512 samples and 50% overlap) was used for spectral estimation. Very low frequency (VLF: 0 to 0.2 Hz), low frequency (LF: 0.20 to 0.75 Hz) and high frequency (HF: 0.75 to 3.0 Hz) (Cerutti et al., 1994; Cerutti et al., 1991) magnitudes were displayed on a logarithmic scale (Log(ms^2)). We analyzed data during the steady-state period, Nadir-1, and recovery. The durations of calculations in the steady state, Nadir-1 and recovery phases were 5, 25 and 30 minutes, respectively. VLF is included for relation to renin-angiotensin system activity (Claydon & Krassioukov, 2008) and metabolic regulatory mechanisms (Millis et al., 2011). LF is used for the evaluations of the sympathetic and parasympathetic activities (Lehrer, 2007) and HF is an indicator of the parasympathetic activity (Laborde et al., 2017).

Calculation of shock index 

We used the shock index (SI) by calculating the ratio of heart rate to systolic blood pressure in order to approximate the hemodynamic status of the animals in the experimental groups. The elevation of SI indicates the fall in left ventricular end-diastolic pressure and blood volume during hemorrhagic shock (Koch et al., 2019).

Arterial blood gas parameters 

The 100 µl of blood samples were taken during the baseline period and at the end of the experiments for the blood gas analysis using an easy blood gas analyzer (Medica, USA).

Measurement of plasma parameters 

At the end of the examinations, 1 ml of the venous blood sample was taken and centrifuged. The plasma was stored at -80 °C. Plasma lactate was measured using an auto-analyzer with commercial reagent (Selectra, China).  

Real-time PCR analysis 

TNF-α and iNOS gene expression were assayed by real-time polymerase chain reaction (PCR). Total RNA of the lung and spleen tissues was extracted by RNA extraction kit TriSolution plus Reagent (GeneMark, Atlanta, GA) according to the manufacturer’s instructions. The quantity and purity of RNA were checked by spectrometer NanoDrop TM (NanodropTM, Thermo Fisher Scientific, Wilmington, DE, USA). Then, RNA was stored at -80 °C until cDNA synthesis. For cDNA synthesis, 2000 ng of RNA was used according to the instructions in the cDNA Fermentas Kit (Fermentas Inc.). The primers were designed based on the DNA sequences, which found in the gene bank Primer-BLAST online program (Ye et al., 2012). Real-time PCR was performed by Applied BioSystems, Step One ™, using the Real Q Plus 2x kit Master Mix Green (Ampliqon Inc); based on the manufacturer’s protocol. The B2M gene was used as a reference in real-time PCR reactions. The real-time PCR system was set within 10 minutes at 95 °C, including 44 cycles (each of 15 seconds at 95 ° C), 60 seconds at 60 °C. Also, a melt curve analysis was used to verify specific amplification. The results were normalized with the B2M cycle threshold (Ct). Finally, fold change expression of TNF-α and iNOS genes were assessed with a 2−∆∆Cq method.

Statistical analysis  

Data are given as mean±SE. Repeated measures ANOVA were used to compare effects groups across the time course of the experiment. Significant effects were followed up with simple ANOVA contrasts and Tukey’s post hoc test, where appropriate. Finally, single time-point measures, namely TNF-α and iNOS, were explored using a parametric one-way ANOVA test with Tukey’s post hoc test. All analysis was performed using the software of SPSS 18. Significance was assumed when p<0.05 and the confidence limits used were the 95% intervals.

Results

Discussion

In this study, we investigated the role of the sub-diaphragmatic vagus nerve on hemodynamic parameters and HRV in long term hemorrhagic shock. Also, blood parameters and the expressions of inflammatory markers in the spleen and lung tissue have been assessed. There were similar alterations in blood pressure during the induction of hemorrhagic shock in the LHS and Vag+LHS group. However, HR did not decrease during the Nadir-1 phase of the Vag+LHS group as seen in the LHS group. MAP and HR did not return entirely during recovery in the Vag+LHS group. Data of HRV indicated increased the VLF, LF, and HF bands in the Nadir-1 phase of the LHS group which decreased to a smaller extent in the Vag+LHS group. All the noted components of HRV were reversed during the recovery phase of the LHS group almost similar to those in the Vag+LHS group. There was a similar metabolic acidosis partially compensated with respiratory system in both groups of LHS and Vag+LHS. Also, the expressions of TNF-α and iNOS increase in the lung were in parallel with those ones in the spleen of the LHS group, whereas, both of the above parameters decreased in aforementioned organs in the Vag+LHS group. Taken together, these findings support a blunted parasympathetic response along the cardiac vagal branch and local differences in the inflammatory impacts in line with the location of the vagotomization.

No alteration was identified in the MAP, HR, HRV indices, and blood gas variables at baselines in all experimental groups. Therefore, all groups enter the study under identical conditions. In addition, these results suggest that the unilateral sub-diaphragmatic vagotomy has no effect on the above parameters during the steady-state period.

During the In-Com phase, MAP remained stable in the LHS and Vag+LHS groups, whereas, HR increased significantly compared with their baselines. Therefore, the constant MAP occurs at the expense of increased HR which can be linked to the sympathetic activity in these groups consistent with other studies (Porter et al., 2009).

In the Nadir-1 phase of the LHS group, MAP was maintained in the lowest range possible in association with decreased HR, similar to other studies in conscious animals subjected to hemorrhagic shock (Troy et al., 2014). However, a significant decrease in MAP despite a small decrease in HR in the vagotomized group of Vag+LHS may be partly linked to the reduction in vascular resistance. It has been reported that cervical vagus nerve stimulation (VNS) after trauma and hemorrhagic shock modulates release of norepinephrine and thereby reduce MAP and HR and increase intestinal blood flow, whereas abdominal vagotomy has an opposite effect (Yagi et al., 2020). In addition, sub-diaphragmatic vagotomy increases the release of epinephrine under stressful conditions (Mravec et al., 2015). Therefore, the high HR during Nadir-1 in the Vag+LHS group may be due to increased catecholamine release following the partial disinhibition of the sympathetic nerve fibers of the adrenal gland. However, neither catecholamines nor its metabolites were measured in this study because it could affect our hemodynamic results during blood withdrawal. On the other hand, both HF and LF increased in the Nadir-1 phase of both LHS and Vag+LHS groups, though these increases in the Vag+LHS group were lower than those in the LHS group. LF band is an indicator of the sympathetic activity (Lehrer, 2007). Besides, it has been reported that during parasympathetic hyper activity, the LF band increases in parallel with HF band. Therefore, the LF band cannot be used as a reliable index for the sympathetic activity in this condition (Saul, 1990). Consequently, the higher LF band in the Nadir-1 phase of the LHS group may be related to the higher HF in this group. Contrary to our results, Payne and her colleges have observed that abdominal vagus nerve manipulation did not evoke any effects on cardiac, respiratory and blood pressure parameters (Payne et al., 2019). The different experimental conditions may produce these dissimilarity results.

The reduction in MAP and HR associated with a significant increase in HF suggests the parasympathetic hyper activity in the Nadir-1 phase of the LHS group. It has been expressed that low pressure baroreceptors in the heart and lungs are activated by losing more than 15–20% of total blood volume, leading to the central inhibition of the sympathetic activity together with the increased parasympathetic activity (Porter et al., 2009). In addition, the increase in the HF band in the Vag+LHS group was significantly lower than that in the LHS group which confirms lowering the parasympathetic activity as a consequence of sub-diaphragmatic vagotomy. Therefore, it might be suggested that the interruption of this pathway through sub-diaphragmatic vagotomy not only interferes with the vagus nerve activity but also may prevent the central weakening the sympathetic activity. As a result, HR did not decrease during the Nadir-1 phase of the Vag+LHS group. Also, during the Nadir-1 phase of the Vag+LHS group, the VLF band was lower than that in the LHS group. It has been indicated that parasympathetic activity is the major determinant of the VLF band (Tripathi, 2011). The results of our study also indicate that the decrease in the parasympathetic component of HF is concomitant with the decrease in VLF in the Vag+LHS group. Coordination of the autonomic nervous system is organized in a hierarchical manner reflective of the evolutionary history of autonomic control (Porges, 2011). Our result may suggested that sub-diaphragmatic vagatomy removes afferent signaling from the organs impacted by hemorrhagic shock, reducing the magnitude of input to the nucleus tractus solitarius and thus attenuating the response in the nucleus ambiguus and dorsal motor vagal nucleus to the challenge. Furthermore, decrease in the VLF band suggests more organ damage in the Vag+LHS group (Ryan et al., 2011).

According to previous study, in case of cessation of blood withdrawal in the class II hemorrhagic shock, MAP and HRV components were returned back to their baselines in both with or without vagotomy groups (Khodadadi et al., 2020). However, the present study showed that with continued blood withdrawal and keeping the MAP in the lowest range; HF component remained high in in the LHS and Vag+LHS groups. These results have suggested that in the Compensatory class of hemorrhagic shock, the parasympathetic activity decreased after cessation of blood withdrawal; in severe classes, the parasympathetic activity remained high as blood withdrawal continued. Therefore, HRV may be a useful predictor of the patient's condition in terms of cessation or continuation of hemorrhage.

During the recovery phase, MAP returned to the baseline value in the LHS group, though it decreased slightly by the end of the experiment. However, MAP in the Vag+LHS, despite the increase in HR, did not return to the baseline level. The HF bands returned to the baselines in the LHS and Vag+LHS group, and the VLF bands were significantly lower than those in their baselines. These results suggest that the difference between the parasympathetic activities in the above two severe hemorrhagic shock group may be reversed after resuscitation. One more possibility could be related to the severity of hemorrhagic shock. We did not measure HR and HRV during the Nadir-2 phase because the infusion of withdrawing blood may affect the results of HR, hemodynamics, and HRV. Therefore, it can be concluded that during a long term hemorrhagic shock, many organs, including the nervous system may be impacted. As a result, HRV per se may not be a valuable indicator for evaluating the conditions of the patients with severe hemorrhagic shock.

At the end of the experiments, the arterial pH, bicarbonate, and BE decreased, and lactate increased in the LHS group. These results indicated metabolic acidosis in the LHS group which occurs as a consequence of delayed resuscitation, disruption of tissue perfusion, and anaerobic metabolism. The decrease in PaCO₂ occurs as a result of a compensatory elevation of ventilation in both hemorrhagic shock groups. In the Vag+LHS group, the metabolic acidosis and the lactate levels were slightly elevated compared to the LHS group, though this difference was not statistically significant. Therefore, it can be proposed that vagotomy has a negative effect on metabolic status during severe hemorrhagic shock in our study.

Our data indicate that vagotomy might lead to the exacerbation of metabolic acidosis. In agreement with our study, it has been reported that vagotomy increases plasma lactate and decreases BE in a thermal injury (Song et al., 2010). However, we have recently indicated that vagotomy exacerbates gas exchange through the blood-gas barrier, and lung tissue inflammation in the class II hemorrhagic shock and have implicated the anti-inflammatory effect of the vagus nerve (Khodadadi et al., 2020). Nevertheless, in the severe hemorrhagic shock in this study, the sub-diaphragmatic vagotomy had the opposite effect. The expressions of TNF-α and iNOS increased in the spleen and lung of the LHS group. TNF-α is mostly released by macrophages leading to inflammation, cell proliferation and differentiation, leukocyte adhesion, and cell apoptosis (McGhan and Jaroszewski, 2012; Zelová & Hošek, 2013). The TNF-α and iNOS expressions in the spleen and lung were suppressed in the Vag+LHS group. Therefore, it may suggest that the anti-inflammatory effect of the vagus nerve depends on the severity of the disease so that it may be masked by the extensive inflammatory reactions in the injured body tissues in severe hemorrhagic shock. In agreement with our conclusion, it has been indicated that laparotomy with or without the removal of the spleen, as an important organ in the cholinergic anti-inflammatory arc, worsen oxidative stress in hemorrhagic shock linked to the serious organ damage (Kilicoglu et al., 2006).

Besides, the inflammatory or anti-inflammatory effects of the vagus nerve are controversial. On one hand, the activation of the afferent vagus nerve may trigger the hypothalamus-pituitary axis, leading to the release of glucocorticoids thereby inhibiting the production of local cytokines (Sternberg, 1997). Furthermore, ACh released from the efferent fibers of the vagus nerve may inhibit the production of inflammatory mediators (Andersson and Tracey, 2012; Hall et al., 2014; Parrish et al., 2008). It has been reported that parasympathetic nervous system activity reduces the inflammatory cytokine expression by inhibiting NF-kB pathway (Guarini et al., 2003). On the other hand, other studies have suggested the inflammatory effect of the vagus nerve (Fuentes et al., 2005). Also, it has been expressed that the effect of the vagus nerve on inflammation depends on the timing of vagal stimulation or denervation, as vagal stimulation at 4 hours after induction of polymicrobial sepsis by cecal ligation and puncture (CLP) does not reduce the lung injury or pulmonary and systemic inflammatory markers (Boland et al., 2011). Nevertheless, the acetylcholinesterase inhibitor attenuates inflammation and septic shock when administered before the induction of CLP (Hofer et al., 2008). Also, vagotomy decreased serum TNF-α at 24 hours before the injection of LPS (Fuentes et al., 2005). Our study may suggest the anti-inflammatory effect of the vagus nerve is reversed in severe tissue damage induced by hemorrhagic shock. However, more studies are needed to disclose the role of the vagus nerve at different times and severities of hemorrhagic shock. Furthermore, there was no correlation between the expressions of TNF-α and iNOS with HRV which is supported by others that there is no association between cytokine levels and HRV in endotoxemia (Kox et al., 2011). In addition, it has been proposed that the threshold of the vagal nerve in regulating immune responses is lower than the threshold needed to alter heart rate (Huston et al., 2007). All these data indicate that HRV may not be a valuable parameter in distinguishing the condition of the patient in severe hemorrhagic shock.

Conclusion

In conclusion, this study indicated an increase in parasympathetic activity obtained from HRV analysis in the Nadir-1 phase of severe hemorrhagic shock. In addition, the parasympathetic activity is reversed during the recovery time. Also, the increases in TNF-α and iNOS expressions in the hemorrhagic shock group were prevented in the vagotomized hemorrhagic shock group which suggests that the effect of the vagus nerve in severe organ damage would be in favor of the increase in inflammation, particularly through afferent signaling from the visceral organs impacted by the hemorrhagic shock.

Abbreviations

LHS: Long term hemorrhagic shock; Vag: Sub-diaphragmatic Vagotomized; Vag+LHS: Sub-diaphragmatic vagotomized with hemorrhagic shock; HRV: Heart rate variability; VLF: Very low frequency; LF: Low frequency; HF: High frequency; MAP: Mean arterial blood pressure; HR: Heart rate; Ach: Acetylcholine; TNF-α: Tumor necrosis factor-α; NF-κβ: Nuclear factor-kappa β; iNOS: Inducible nitric oxide synthase; COX-2: Cyclooxygenase 2; MDA: Malondialdehyde; In-Com: Initial compensatory; De-Com: De-compensatory; SS: Steady state; ABG: Arterial blood gas analyzing; BP: Blood pressure; SI: Shock index; PCR: Polymerase chain reaction; PaO2: Arterial venous oxygen pressure; PaCO2: Arterial carbon dioxide pressure; VNS: Vagus nerve stimulation; CLP: Cecal ligation and puncture; Ct: Cycle threshold.

Declarations

Acknowledgements 

We would like to appreciate Dr Iman Jamhiri for technical assistance in PCR measurements.

Author contributions 

F.Kho and A.T performed experiments; F.Ket and F.Kho analyzed data; F.Ket, F.Kho, A.B and A.T interpreted results of the experiments; F.Kho prepared figures; F.Kho and F.Ket drafted the manuscript; F.Ket , A.B and G. L edited and revised manuscript; all authors have read and approved final version of the manuscript.  

Funding 

This work was supported by the Research Council of University of Shiraz [grant number SU-9330208]; and the Research Council of Shiraz University of Medical Sciences [grant number 96-01-01-14378] as a part of work of acquiring a Ph.D degree in physiology by F. Khodadadi. The authors confirm that none of these organizations had a role in the design of the study, data collection, data analysis, interpretation of data, or in writing the manuscript.

Availability of data and materials

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

All procedures in this study were approved by the Center for Comparative and Experimental Medicine and the Ethical Committee of Animal Care at Shiraz University of Medical Sciences, Shiraz, Iran. The experiments were conducted in accordance with the declaration of Helsinki (approval code no: IR.SUMS.MED.REC.1396.s203). All methods are reported in accordance with ARRIVE guidelines (Du Sert et al., 2020).

Consent for publication

Not applicable.

Competing interests 

The authors declare no competing interests.

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