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.