Methylene blue therapy versus standard treatment for acute-phase septic shock: a pilot randomized controlled trial

Methylene blue therapy versus standard treatment for acute-phase septic shock: a pilot randomized controlled trial | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Methylene blue therapy versus standard treatment for acute-phase septic shock: a pilot randomized controlled trial Fabio Luis-Silva, Mayra Gonçalves Menegueti, Leandro Moreira Peres, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3971422/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Methylene blue (MB) has been used to increase blood pressure in patients with septic shock by acting on guanylate cyclase and nitric oxide synthase (NOS). Objective To determine whether the administration of MB to patients in the initial phase of septic shock leads to a reduction in the use of vasopressors compared to that in the control group. Methods This was a 1:1 randomized clinical trial of two groups (methylene blue and control). We used MB after fluid replacement, vasopressors and antibiotic therapy. Patients received a loading dose of MB (3 mg/kg) and maintenance (0.5 mg/kg/h) for 48 hours. Vasopressor doses, laboratory test results, inflammatory and anti-inflammatory cytokine levels, and hemodynamic monitoring were recorded before the infusion of MB (T1) and after 20 minutes (T2), 2 hours (T3), 24 hours (T4), 48 hours after the infusion started (T5) and 24 hours after weaning (T6). Results Methylene blue therapy started within 72 hours of septic shock. The methylene blue group showed an immediate reduction in NOR dosage, earlier reduction in VAS dosage, and higher IL-10 levels compared to the control group. Integrative network analysis highlighted NO and IL-10's roles in coordinating correlations with "Hemodynamic Monitoring" in the control and methylene blue groups, respectively. Conclusion Early methylene blue (MB) administration alongside standard septic shock treatment reduces vasopressor doses, possibly involving nitric oxide (NO) mechanisms. A possible mechanism of action may involve modulation of inflammatory and anti-inflammatory mediators, enhancing immune response. However, larger and longer studies are needed for validation. Trial registration: Clinical registration: https://ensaiosclinicos.gov.br/rg/RBR-96584w4 . Methylene Blue Septic Shock Lactate Nitric Oxide Cytokines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Septic shock is characterized by changes in the hemodynamic profile of patients, among which are increased cardiac output and persistent systemic arterial hypotension secondary to arterial vasodilation; this condition is associated with an intense systemic inflammatory reaction and is the main cause of morbidity and mortality in patients admitted to intensive care units (ICUs) [ 1 , 2 ], while mortality ranges between 38 and 46.5% [ 3 ]; however, epidemiological data are scarce and practically nonexistent in developing countries, and a provisional extrapolation of data from high-income countries suggests global estimates of 5.3 million deaths annually [ 4 ], which are serious public health problems. A diagnosis of septic shock occurs in patients with a confirmed or presumed focus of infection associated with a mean arterial pressure (MAP) ≤ 65 mmHg and a lactate concentration ≥ 2.0 mmol/L after adequate volume replacement [ 5 ]. Treatment includes fluid replacement, infusion of vasopressors, and administration of antibiotics within the first hour [ 1 ]; these treatments are associated with low-dose corticosteroids in refractory cases [ 6 ], but mortality remains high, especially in developing countries. Studying new medications that help maintain hemodynamic stability until the antibiotic acts and combats the infectious focus is necessary [ 7 ]. Methylene blue (MB), a nonselective inhibitor of soluble guanylate cyclase (SGC) and nitric oxide synthase (NOS), is a heterocyclic aromatic compound from the phenothiazine class that has been used since the 19th century and has had proven hemodynamic effects since 1976 [ 8 ]; additionally, MB has been used safely, with few side effects when it is used at adequate doses. The safety of intravenous administration and dosages of 1 to 3 mg/kg have been previously reported, and the authors state that excessive doses of MB result in adverse effects on visceral tissue perfusion. Doses greater than 40 mg/kg are lethal [ 1 , 9 ]. Several studies have proposed that MB can treat circulatory shock secondary to vasoplegia via nitric oxide (NO) [ 7 , 8 , 10 – 15 ]. In a rat model of endotoxemia induced by lipopolysaccharide (LPS) and treated with MB, complete prevention of leukocyte rolling and adhesion to the endothelium was observed, resulting in a consistent systemic blood pressure response and a decreased need for vasoactive medications [ 16 ]. In animal endotoxemia and in patients with septic shock, decreasing plasma levels of stable nitric oxide (NO) metabolites, nitrites, nitrates, and cGMP were demonstrated [ 1 , 10 , 12 ]. Although the medical literature does not include robust studies on the use of MB in septic shock, even with a small sample, the results are promising regarding the association of MB with conventional treatment in these patients [ 7 , 10 , 12 ]. In theory, the inhibition of excessive NO could act favorably, preventing systemic vasodilation and reducing microvascular injury in septic individuals. Another theory is that MB improves mitochondrial function and adenosine triphosphate (ATP) production in cells, helping to reverse cardiovascular dysfunction associated with septic shock [ 17 ]. Some studies have used MB to reverse vasoplegia during the postoperative period after major surgery [ 18 – 22 ], during anaphylactic shock, during refractory shock in traumatized patients and during the postoperative period after liver transplantation [ 23 – 25 ]. Other studies have shown that reducing vasopressors and maintaining adequate MAP levels are beneficial for patients with septic shock [ 14 , 26 – 28 ], but these are small studies and lack robust evidence for this approach. A retrospective cohort study [ 29 ] also showed the benefit of MB in different types of circulatory shock (septic, cardiogenic and vasoplegic), with a reduced need for noradrenaline and reduced mortality after 28 days of observation; however, these findings highlight the importance of performing a prospective and randomized study. A randomized clinical trial in patients with septic shock showing that the administration of MB within 24 hours reduced the time to vasopressor discontinuation did not evaluate possible hemodynamic changes, inflammatory mediators, or the dosage of nitrite or nitrate [ 12 ]. The use of MB was also described in two randomized clinical trials evaluating its use in the treatment of septic shock at an early stage (less than 24 hours after diagnosis), which demonstrated a reduction in and early weaning of vasopressors [ 30 , 31 ]. However, these studies did not use invasive hemodynamic monitoring or the measurement of inflammatory or anti-inflammatory mediators or NO for 72 hours, as in the present study. Studies have shown the beneficial effects of MB in patients with septic shock [ 2 , 12 , 32 ]; however, studies involving patients in the acute phase are rare [ 7 , 10 , 12 ]. Our hypothesis is that MB contributes to reducing the infusion of vasopressors, improving tissue perfusion and delaying mitochondrial death induced by nitric oxide if administered in the first 48 hours of septic shock. Therefore, the aim of the present study was to determine whether the administration of MB to patients in the initial phase of septic shock leads to a reduction in the use of vasopressors compared to that in the control group. Material and Methods Study design, population and sampling This was a pilot randomized clinical trial conducted in the ICU of a tertiary university hospital from January 2019 to August 2023. All the legal guardians of the included patients agreed and signed the free and informed consent form. The Ethics Committee of the Hospital das Clínicas of the Faculty of Medicine of Ribeirão Preto approved this protocol–number: 562/2017, version: 2/2016.Blinding was not possible since MB leaves body fluids with a bluish-green color are easily identified upon use. To minimize possible biases, the professional responsible for randomization had no access to patient clinical records, and the researcher did not know the patient group when performing hemodynamic monitoring for data collection. Figure 1 summarizes the study population and study design. Eighty patients were enrolled upon signing the informed consent form by their next of kin and were randomly allocated into two groups according to the previously published protocol [ 13 ]. After withdrawing based on clinical status (n = 48), a total of 42 patients were included in the present study; 23 patients were allocated to the control group, and 19 were randomized to the MB group. No previous studies in the literature have used continuous-dose MB for 48 hours to support the sample size calculation. Therefore, in the present investigation, the sample size was defined according to the guidelines proposed by Sandvik et al. [ 33 ] for pilot clinical trials, which recommended the inclusion of 20 patients in each study arm. After measuring and collecting initial exams, patients in the MB group received conventional treatment and MB at a dose of 3 mg/kg in 20 min and then 0.5 mg/kg/h for 48 hours, and patients in the control group received conventional treatment. Conventional treatment included fluid replacement, vasopressor drugs (noradrenaline and vasopressin), hydrocortisone (200 mg/day) and antibiotics within the first hour. After completion of the protocol, patients were followed up for 30 days to assess the outcome, discharge status, or death status. After randomization, the following procedures were performed: weight measurement using a bed scale (Styker® brand), catheter insertion in the femoral vein (VolumeView System®), and central venous access in the internal jugular or subclavian vein (Presep®) for monitoring with the EV1000® platform (Edwards Lifesciences Corporation®). The SAPS3 (Simplified Acute Physiology Score 3) and APACHE II (Acute Physiology and Chronic Health Evaluation) prognostic scores were calculated for the first 24 hours after admission, and the daily SOFA score was calculated for the four days of the protocol. Table 1 summarizes the demographic and clinical characteristics of the study population. Considering the patients with positive cultures, empirical antibiotics were started less than three hours after the diagnosis of septic shock, providing adequate coverage in 43.5% of the control group and 21.1% of the MB group (p < 0.1240). Table 1 Demographic and clinical features of the study population. Variables Group Control (n = 23) MB (n = 19) p value* Sex (Male) – n (%) 16 (70) 14 (74) 0.7700 Age (Years) – Mean 42.7 ± 17.7 51.5 ± 14.3 0.0876 Hypertension – n (%) 7 (30) 6 (32) 0.9300 Diabetes – n (%) 4 (17) 3 (16) 0.8900 Acute kidney injury – n (%) 14 (61) 15 (79) 0.2100 Hemodialysis – (%) 6 (27) 2 (11) 0.2000 SAPS3 – Mean ± SD 58.2 ± 9.8 78.1 ± 11.4 < 0.0001 Death Risk – Mean ± SD 47.1 ± 20.6 82.0 ± 13.5 < 0.0001 APACHE II – Mean ± SD 21.7 ± 9.6 39.6 ± 6.8 < 0.0001 SOFA – Mean ± SD T1 (Baseline) 9.0 ± 2.7 12.1 ± 2.8 0.0010 T4 (24 hours) 8.2 ± 3.1 11.2 ± 3.2 0.0040 T5 (48 hours) 7.3 ± 3.3 10.8 ± 3.4 0.0020 T6 (72 hours) 7.2 ± 3.7 10.5 ± 3.4 0.0040 Sites of infection Abdomen – n (%) 5 (21.7) 7 (36.8) 0.3000 Lung – n (%) 15 (65.1) 6 (31.6) 0.0300 Urinary tract – n (%) 1 (4.4) 3 (15.8) 0.2450 CRBSI – n (%) 1 (4.4) 0 (0) 0.3280 Other – n (%) 1 (4.4) 3 (15.8) 0.2450 Positive culture – n (%) 13 (56.5) 13 (68.4) 0.4390 Effectiveness of empiric antibiotic – (%) 43.5% 21.1% 0.1240 Days of antibiotic use – Mean ± SD 18.9 ± 20.0 16.4 ± 12.5 0.6386 Shock time at baseline (hour) – Mean ± SD 44.5 ± 24.5 33.3 ± 12.3 0.0738 Fluid balance (mL in 72 hours) – Mean ± SD 1,416 ± 3,158 2,871 ± 3,163 0.1453 30 days outcome (Death) – n (%) 14 (61) 9 (47) 0.3800 * MB: Methylene blue; SD: standard deviation; CRBSI – Catheter-Related Blood Stream Infection. Chi-square test was used for categorical data and Student’s test employed for continuous variables comparisons. Significant differences are underscored in italic. Hemodynamic and Vasopressor Drug Monitoring After calibrating the EV1000® platform, the mean arterial pressure (MAP), cardiac output (CO), cardiac index (CI), heart rate (HR), systemic vascular resistance index (SVRI), central venous pressure (CVP), stroke volume index (SVI), stroke volume variation (SVV), global ejection fraction (GEF), pulmonary vascular permeability index (PVPI), global end diastolic volume index (GEDVI) and extravascular lung water index (ELWI) were measured. Measurements of central venous oxygen saturation (ScVO 2 ), arterial blood gas, serum lactate, bilirubin, complete blood count, sodium, potassium, urea, and creatinine were also performed. The oxygen delivery index (DO 2 I), oxygen consumption index (VO 2 I), and oxygen extraction rate (O 2 ER) were calculated. Vasopressor drug monitoring (norepinephrine/NOR and vasopressin/VAS) was carried out daily by a physician according to a standardized institutional protocol. Laboratory analysis of immunological features To evaluate the immunological parameters, serial whole-blood sampling was performed along the study timeline (T1 to T6) using EDTA and heparin. Samples were subjected to centrifugation at 3,500 rpm for 10 minutes at 16°C, and plasma aliquots were stored at -80°C until processing. Soluble immune mediator (interleukin 8 [IL-8/CXCL8], interleukin 6 [IL-6], tumor necrosis factor alpha [TNF-α], and interleukin 10 [IL-10]) levels were measured in EDTA-treated samples according to the manufacturer’s instructions (R&D Systems), and nitric oxide (NO 3 ) levels were measured in heparin-treated samples via chemiluminescence according to the manufacturer’s instructions (Sievers NO Analizer). Statistical analysis The chi-square test was used to verify the associations of qualitative variables between the MB and control groups. Student’s t test was used to analyze quantitative clinical variables. The comparison of immunological features between the MB and control groups at each timepoint was performed by the Mann‒Whitney test. The Spearman rank test was used to perform cross-correlation analysis of vasopressor drugs and hemodynamic and immunological variables (attributes) among all timepoints. GraphPad Prism software was used for all the statistical analyses. In all cases, p < 0.05 was considered to indicate statistical significance. Significant correlations were used to construct integrative networks. Integrative networks were built using the systems biology approach of the Cytoscape open-source platform (available at https://cytoscape.org ) based on the “r” scores of significant correlations. The networks were assembled using a cluster layout with nodes used to represent each variable, and connecting lines were employed to identify positive (continuous line) and negative (dashed line) correlations. The node sizes are proportional to the number of correlations between parameters. Line thickness illustrates the correlation strength, ranging from weak/moderate (“r” scores from |0.1 to 0.6|, thin lines) to strong correlations (“r” scores from ≥ |0.7|, thick lines). The red line illustrates the correlations between immunological features and vasopressor drugs. Correlation matrices were assembled using the “corrplot” package of R software (Project for Statistical Computing Version 3.0.1). Microsoft Excel and Prism GraphPad software were used to create the graphics. Results MAP and vasopressor doses along the kinetic timeline in the MB and control groups The analysis of MAP and vasopressor doses along the kinetic timeline was assessed by comparing pairs of adjacent timepoints. Figure 2 shows the median MAP values along with the NOR and VAS scores along the kinetic timelines in the MB and control groups. The data analysis did not reveal significant differences in the MAP during the kinetics follow-up. The NOR dose was markedly lower in the MB test at T2 than at T1, with a continuous decrease toward T5, while in the control group, a decrease in NOR was observed later at T4 than at T3. A significant difference in NOR dose between the MB and control groups was observed at T3. The analysis of VAS scores demonstrated early withdrawal in the MB group at T4, while in the control group, VAS scores were withdrawn later at T5. A significant difference in VAS score between the MB and control groups was observed at T4. (Fig. 2 ). Analysis of several hemodynamic variables (CO, CI, HR, SVRI, CVP, SVI, SVV, GEF, PVPI, GEDVI and ELWI) along the kinetics timeline (T1 to T6) did not reveal significant differences (data not shown). A detailed description of the median values of MAP and vasopressor doses along the kinetic timeline is provided in Supplementary Table 1. Kinetic timeline of serum lactate concentration, DO 2 I, VO 2 I and O 2 ER × CO in the MB and control groups The analysis of hemodynamic variables (serum lactate concentration, DO 2 I, VO 2 I and O 2 ER × CO) was assessed along the kinetic timeline by comparing pairs of adjacent timepoints. Figure 3 shows the median values of the serum lactate concentration, DO 2 I, VO 2 I and O 2 ER × CO. Data analysis demonstrated that while the serum lactate concentration decreased in the MB group at T2 compared to T1, no differences were observed between adjacent timepoints in the control group. The analysis of DO 2 I demonstrated an early increase in MB at T2 and T3 and a later increase at T5 in the control group, leading to differences between the MB and control groups. No significant differences were observed for VO 2 I or O 2 ER × CO along the kinetics timeline. The analysis of fold changes further corroborated these findings (Fig. 3 ). A detailed description of the median values of the serum lactate concentration, DO 2 I, VO 2 I and O 2 ER × CO concentration along the kinetic timeline is provided in Supplementary Table 1. Changes in plasma immune mediator and nitric oxide levels in the MB and control groups Figure 4 presents the overall profile of soluble plasma immune mediators and NO in the MB and control groups. These immune mediators were chosen because they are related to the inflammatory response in sepsis, thus making it possible to evaluate whether treatment with MB alters the inflammatory response. The analysis of immune mediators and NO was assessed along the kinetic timeline by comparing pairs of adjacent timepoints. The data analysis demonstrated an increase in CXCL8 in the MBs at T6 compared to T5 and a decrease in the control group at T5 compared to T4. The data showed an increase in IL-6 in the control group at T2, with a progressive decrease toward T5 and a decrease in MB at T4 compared to T3. Compared with those in the control group, TNF-α in the MB at T5 were lower than those in T4, and lower levels were detected in the MB at T5 and T6. The analysis of IL-10 showed lower levels in the control group at T2 than at T1. Higher levels of IL-10 were observed in the MBs at T1 and T6 than in the control group. NO was elevated at T3 in the control group and progressively decreased toward T5. In the MB group, the levels of NO were greater than those in the control group at T4 and T5 and displayed a progressive decrease toward T6. The analysis of fold changes corroborated these findings. A detailed description of the median values of plasma immune mediators and nitric oxide concentrations along the kinetic timeline is provided in Supplementary Table 2. Integrative networks of vasopressor drugs and hemodynamic and immunological variables in the MB and control groups Figure 5 displays the integrative network of vasopressor drugs and hemodynamic and immunological variables in the MB and control groups. Integrative networks were built using systems biology approaches with a cluster layout with nodes used to represent each variable and connecting lines used to identify positive and negative correlations between pairs of attributes. The node sizes are proportional to the number of correlations. Despite the similar numbers of correlations observed in the MB and control groups (n = 48 and n = 49, respectively), intracluster analysis demonstrated greater contributions of the “immune mediators” (32% vs 28%) and “MAP;NOR;VAS” clusters in the MB (19% vs 16%) cohort than in the control group. Conversely, more correlations within the “Hemodynamic Monitoring” (56% vs 49%) cluster were observed in the Control group than in the MB group. Notably, while NOR and VAS scores were directly correlated with NO in the control group, they were directly correlated with IL-10 in the MB group (Fig. 5 , red connecting lines). Overall, the integrative network analysis demonstrated that, while NO represents a key attribute orchestrating the correlation with the “Hemodynamic Monitoring” cluster in the control group, IL-10 plays a pivotal role in coordinating the correlation with the “Hemodynamic Monitoring” cluster (Fig. 5 , thick connecting lines). This was more evident for the ScVO 2 , O 2 ER and O 2 ER × CO attributes. Taken together, these findings indicate that the underlying mechanism through which MBs impact hemodynamic features in septic shock may include not only the suppression of NO activity but also the involvement of other events mediated by IL-10. Detailed correlogram data supporting the integrative network are provided in Supplementary Fig. 1. Discussion The present study used MB to reduce NO-mediated vasoplegia, which can minimize the use of vasopressors, in addition to being a low-cost medication, without side effects and being easily accessible in health units [ 12 , 34 ]. We started MB in the first 72 hours of septic shock and observed a reduction in the dose of NOR immediately after the start of the infusion compared to that in the control group, where this reduction occurred only after 24 hours. Similar findings were described in a randomized clinical trial that demonstrated that early use of MB reduced the duration of vasopressor use compared to that in the control group, and most importantly, no serious adverse effects were detected. The authors further suggest that MB should not be used as rescue therapy but rather as adjuvant therapy in the early stages of septic shock. [ 12 , 34 ]. Due to its safety profile, greater availability and lower cost than other catecholamine-sparing agents [ 35 ], MB could emerge as a viable therapy within a multimodal strategy to maintain MAP and improve tissue perfusion. Furthermore, MB contributes to reducing the use of high-dose vasopressors [ 21 , 23 , 24 , 29 – 31 , 36 – 39 ], which was also found in our study. When evaluating weaning from VAS, we observed early withdrawal in the MB group within 24 hours, while in the control group, VAS withdrawal was observed within 48 hours. The study of Preiser et al.[ 25 ] investigated the intravenous administration of MB at a single dose of 2 mg/kg in patients with septic shock and demonstrated an increase in MAP and IRVS, but these hemodynamic improvements were not sustained, suggesting that longer MB administration deserves further investigation. In our study, we performed a continuous infusion of MB for 48 hours and observed a decrease in the use of vasopressors within 2 hours that was maintained during the period of MB infusion. Perhaps this time is still short since many patients in the present study still needed the use of vasopressors, so the shock had not yet been fully resolved. Furthermore, there was a greater need for vasopressors after MB suspension. We speculate that in septic shock, a longer time is needed for complete hemodynamic restoration since the effectiveness of the antimicrobial agent is important and is linked to access to the culture results. A randomized clinical trial in patients with septic shock showed that the administration of MB within 24 hours reduced the duration of vasopressor suspension but did not evaluate possible hemodynamic changes, inflammatory mediators, nitrites or nit dosages [ 9 ]. In the present study, we observed that the dosage of nitrate (a NO metabolite) in the MB group was greater than that in the control group during the first 48 hours (T4). This can be explained by the fact that MB, by blocking guanylate cyclase, increases NO, but it remains inactive because it depends on this enzyme. This high dosage of nitrate in the MB group may have been beneficial since NO deficiency can lead to endothelial dysfunction, increased insulin resistance and impairment of the immune system [ 40 ]. Another important aspect of MB is its antioxidant properties, which eliminate reactive oxygen species (ROS) independently of cGMP, helping to protect cells against oxidative damage or even helping to improve mitochondrial function [ 17 ]. However, few robust studies have evaluated the dosage of cytokines in patients who use MB for septic shock. Memis et al. [ 31 ] evaluated the dosage of TNF-α, IL-1, IL-2, IL-6 and CXCL8 and found no changes in serum levels during 6 hours of MB use. We speculate that these results differ from our study, as prolonged use of MB could trigger more sustained effects on inflammatory and anti-inflammatory responses. In the present study, we also observed an increase in CXCL8 in the MB group at T2 to T3, which may have contributed to improved targeting of granulocytes, benefiting the acute phase of septic shock [ 41 ]. In the control group, there was a progressive reduction from T1 to T5; however, there was an increase from T4 to T6, at which point we already expected a reduction due to the control of the inflammatory and infectious process. In the control group, the TNF-α concentration progressively increased, mainly after T3, and remained elevated until T6, which may indicate an excessive response to the infection, even 72 hours after the start of treatment. In the MB group, there was stabilization between T1 and T2, with a decrease in T3 and a much less pronounced increase in T4, with a subsequent decrease until T5, which increased again after withdrawal of the maintenance dose of MB in T6. Although patients in the MB group had more severe illnesses according to prognostic indices, we noticed that in the MB group, there was a lower increase in the level of this proinflammatory cytokine. TNF-α plays an important role in septic shock, signaling the path that defense cells must follow, activating the immune system and increasing the antigen-specific response; however, excessive TNF-α activation is harmful, leading to cell death [ 36 ]. IL-10, an anti-inflammatory cytokine, was expressed at lower levels from T1 to T4 in the control group than in the MB group, with increases in T5 and T6 in the MB group compared to the control group. This elevation observed in the MB group may play a role in maintaining homeostasis between inflammatory and anti-inflammatory mediators. In this study, we also analyzed the integrative networks of vasopressor drugs and hemodynamic and immunological variables in the MB and control groups. The data analysis demonstrated that while NOR and VAS scores were directly correlated with NO in the control group, they were directly correlated with IL-10 in the MB group. These findings demonstrate that the mechanisms underlying the effect of MB on hemodynamic characteristics in septic shock may rely not only on blocking the action of NO but also on other events mediated by IL-10, which may be one of the possibilities for hemodynamic improvement, independent of action and serum nitrate levels. In addition to the vasoconstrictor effect, norepinephrine and vasopressin can also act by decreasing the production of nitric oxide [ 42 , 43 ]. Thus, in the control group, in which MB was not used, vasopressors showed a direct and strong correlation with nitrate. However, when we used MBs, there was no correlation between the use of vasopressors and nitrate. As mentioned above, when guanylate cyclase is blocked, NO levels remain elevated but are inactive. Furthermore, guanylate cyclase, when inactivated, prevents the conversion of GTP into cGMP, which, in addition to reducing vasodilation, also facilitates the action of noradrenaline via cAMP since the cGMP pathway, when little activated, releases cAMP [ 44 ]. Furthermore, in the MB group, vasopressors showed a strong and direct correlation with IL-10, which also affects nitrate reduction [ 45 , 46 ]. Finally, we also observed that patients in the MB group had more severe disease according to the SOFA, SAPS3 and APACHE II scores. However, patients in the MB group used fewer days of vasopressors and had a mortality rate of 47.37% versus 60.87% in the control group. This lower mortality may be related to a balance between inflammatory and anti-inflammatory mediators, which is associated with the theory that describes an improvement in mitochondrial function and the production of adenosine triphosphate (ATP) in cells, helping to reverse cardiovascular dysfunction associated with septic shock [ 17 ]. Some limitations must be recognized. First, this was a single-center study; second, it was an unblinded study, as highlighted in the methodology section. Conclusion Early methylene blue (MB) administration alongside standard septic shock treatment reduces vasopressor doses, possibly involving nitric oxide (NO) mechanisms. A possible mechanism of action may involve modulation of inflammatory and anti-inflammatory mediators, enhancing immune response. However, larger and longer studies are needed for validation. Abbreviations APACHE II = Acute Physiology and Chronic Health Evaluation; ATP = Adenosina Triphosphate; MB = Methylene Blue; CI = Cardiac Index; cNOS = Constitutive Nitric Oxide Synthase; CO = Cardiac Output; SGC = Soluble Guanylate Cyclase; DO 2 = Oxygen Delivery; GEF = Global Ejection Fraction; HR = Heart Rate; PEWI = Pulmonary Extravascular Water Index; ICU = Intensive Care Unit; GEDVI = Global End-Diastolic Volume Index; iNOS = Induced Nitric Oxide Synthase; PVPI = Pulmonary Vascular Permeability Index; SVRI = Systemic Vascular Resistance Index; SVI = Systolic Volume Index; MAP = Mean Arterial Pressure; NO = Nitric Oxide; NOS = Nitric Oxide Synthase; CVP = Central Venous Pressure; SAPS3 = Simplified Acute Physiology Score; SOFA = Sequential Organ Failure Assessment; ScVO 2 = Central Venous Oxygen Saturation; SVV = Systolic Volume Variation; VO 2 : Oxygen Consumption. Declarations Acknowledgments We would like to thank everyone involved in the implementation of this project, especially the medical and nursing staff of the Hospital das Clínicas Ribeirão Preto University of São Paulo. MAM thank the Fundação de Apoio ao Ensino, Pesquisa e Assistência (FAEPA) of the Clinics Hospital of Ribeirão Preto and FAPESP for the grant (#2018/26553-0) and the research fellowships program (#2021/01195-6). OAMF received PQ fellowships from CNPq. OAMF is a research fellow from the Universidade do Estado do Amazonas-UEA (PROVISIT No. 005/2023-PROPESP/UEA). Author contributions FLS and MAM wrote the paper; FLS, MGM, LMP, CRS, BCPM, MDD, BLM, FBS and GHMR were responsible for data collection for the study; MAM, OAMF, JPBS and IACR prepared tables and figures; MAM, MGM and OAMF were responsible for interpretation and statistical analysis; MCJ, FM and CB were responsible to laboratorial analysis and MAM, MGM, PRBE and ABF reviewed the manuscript. Individual patient data will remain confidential. Funding We received funding from FAPESP through the grant (#2018/26553-0). Availability of data and materials All statistical analysis were carried out using GraphPad Prism software, version8.0, (San Diego, CA, USA). All participants received a numerical identification from the study itself. Personal data will be stored in the hospital’s digital system, password protected to ensure confidentiality. Any modifications or adverse events related to the protocol were communicated to REBEC.gov and to the Research Ethics Committee and Clinical Research Unit of Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto by the researcher in charge. All principal investigators have access to the final trial dataset. Datasets used and/ or analyzed during the current study are available from the corresponding author upon reasonable request. Ethical approval and consent to participate This clinical trial is registered at REBEC (https://ensaiosclinicos.gov.br/rg/RBR-96584w4) on June 12, 2021. The Ethics Committee of the Hospital das Clínicas of the Faculty of Medicine of Ribeirão Preto approved this protocol–number: 562/2017, version: 2/2016. Informed consent Terms of free and informed consent were collected from all study participants through their legal guardians. Conflict of interest statement The authors declare that there are no conflict of interest. References Juffermans NP, Vervloet MG, Daemen-Gubbels CR, Binnekade JM, de Jong M, Groeneveld AJ. A dose-finding s tudy of methylene blue to inhibit nitric oxide actions in the hemodynamics of human septic shock . Nitric Oxide. 2010; doi: https://doi.org/10.1016/j.niox.2010.01.006 Zhao CC, Zhai YJ, Hu ZJ, Huo Y, Li ZQ, Zhu GJ. Efficacy and safety of methylene blue in patients with vasodilatory shock: A systematic review and meta-analysis. Front Med. 2022; doi: https://doi.org/10.3389/fmed.2022.950596 Reinhart K, Daniels R, Kissoon N, Machado FR, Schachter RD, Finfer S. Recognizing Sepsis as a Global Health Priority - A WHO Resolution . N Engl J Med. 2017; doi: 10.1056/NEJMp1707170 Fleischmann C, Scherag A, Adhikari NK, Hartog CS, Tsaganos T, Schlattmann P, et al. Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations . Am J Respir Crit Care Med. 2016.; doi: https://doi.org/10.1164/rccm.201504-0781OC Shankar-Hari M, Phillips GS, Levy ML, Seymour CW, Liu VX, Deutschman CS, et al. Developing a New Definition and Assessing New Clinical Criteria for Septic Shock: For the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016; doi: 10.1001/jama.2016.0289 Evans L, Rhodes A, Alhazzani W, Antonelli M, Coopersmith CM, French C, et al. Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock 2021. Crit Care Med. 2021; doi: 10.1097/CCM.0000000000005337 Luis-Silva F, Menegueti MG, Sato L, Peres LM, dos Reis Sepeda C, Petroski-Moraes BC, et al. Effect of methylene blue on hemodynamic response in the early phase of septic shock: A case series. Medicine (Baltimore). 2023; https://doi.org/10.1097%2FMD.0000000000032743 Evora PR, Alves Junior L, Ferreira C. A., Menardi AC, Bassetto S, Rodrigues AJ. Twenty years of vasoplegic syndrome treatment in heart surgery. Methylene blue revised. Rev Bras Cir Cardiovasc. 2015; doi: https://doi.org/10.5935/1678-9741.20140115 Aguilar Arzápalo MF, López Avendaño VG, Escalante Castillo A, Góngora Mukul JJ, Franco Herrera B, Cetina Cámara M A. Eficacia del azul de metileno como coadyuvante en el tratamiento de pacientes con choque séptico. Rev Asoc Mex Med Cirt Ter Int. 2016;30(2):102-110. Puntillo F, Giglio M, Pasqualucci A, Brienza N, Paladini A, Varrassi G. Vasopressor-sparing action of methylene blue in severe sepsis and shock: a narrative review. Advances in Therapy. 2020; doi: https://doi.org/10.1007/s12325-020-01422-x Ismail R, Awad H, Allam R, Youssef O, Ibrahim M, Shehata B. Methylene blue versus vasopressin analog for refractory septic shock in the preterm neonate: A randomized controlled trial. J Neonatal Perinatal Med. 2022; doi: 10.3233/NPM-210824 Ibarra-Estrada M, Kattan E, Aguilera-González P, Sandoval-Plascencia L, Rico-Jauregui U, Gómez-Partida CA. Early adjunctive methylene blue in patients with septic shock: a randomized controlled trial . Crit Care. 2023; doi: https://doi.org/10.1186/s13054-023-04397-7 Luis-Silva F, Luis-Silva F, Menegueti MG, dos Reis Sepeda C, Petroski-Moraes BC, Sato L, Peres LM, Auxiliadora-Martins M. Effect of methylene blue on hemodynamic and metabolic response in septic shock patients . Medicine (Baltimore). 2022; doi: https://doi.org/10.1097%2FMD.0000000000028599 Graudins A, Lee HM, Druda D. Calcium channel antagonist and beta-blocker overdose: antidotes and adjunct therapies . Br J Clin Pharmacol. 2016; doi: https://doi.org/10.1111/bcp.12763 McRobb CM, Holt DW. Methylene blue-induced methemoglobinemia during cardiopulmonary bypass? A case report and literature review . J Extra Corpor Technol. 2008;40(3):206-14. Heger A, Römisch J, Svae TE. A biochemical comparison of a pharmaceutically licensed coagulation active plasma (Octaplas) with a universally applicable development product (Uniplas) and single-donor FFPs subjected to methylene-blue dye and white-light treatment. Transfus Apher Sci. 2006; doi: https://doi.org/10.1016/j.transci.2006.08.008 Rojas JC, Bruchey AK, Gonzalez-Lima F. Neurometabolic mechanisms for memory enhancement and neuroprotection of methylene blue . Prog Neurobiol. 2012; doi: https://doi.org/10.1016/j.pneurobio.2011.10.007 Kim JH, Kim JH, Ku NS, Kim YJ, Kim HB, Seok H, Lee DG,... Park DW. Korean Registry for Improving Sepsis Survival (KISS): Protocol for a Multicenter Cohort of Adult Patients with Sepsis or Septic Shock. Infect Chemot. 2020; doi: https://doi.org/10.3947/ic.2020.52.1.31 Paciullo CA, Methylene blue for the treatment of septic shock . Pharmacotherapy, 2010; doi: https://doi.org/10.1592/phco.30.7.702 Annane D, Annane D, Aegerter P, Jars-Guincestre MC, Guidet B. Current epidemiology of septic shock: the CUB-Réa Network . Am J Respir Crit Care Med. 2003; doi: https://doi.org/10.1164/rccm.2201087 Schneider F, Lutun PH, Hasselmann M, Stoclet JC, Tempe JD. Methylene blue increases systemic vascular resistance in human septic shock. Preliminary observations. IntCare Med. 1992; doi: https://doi.org/10.1007/BF01706481 Rivers E, Nguyen B, Havstad S, Ressler J, Muzzin A, Knoblich B,...Tomlanovich M. Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med. 2001; doi: 10.1056/NEJMoa010307 Daemen-Gubbels CR, Groeneveld PH, Groeneveld AJ, van Kamp GJ, Bronsveld W, Thijs LG. Methylene blue increases myocardial function in septic shock. Crit Care Med. 1995; 23(8):1363-70. Gachot B, Bedos, JP, Veber B, Wolff M, Regnier B. Short-term effects of methylene blue on hemodynamics and gas exchange in humans with septic shock. Intensive Care Med. 1995; doi: https://doi.org/10.1007/BF01700666 Preiser JC, Lejeune P, Roman A, Carlier E, De Backer D, Leeman M,...Vincent JL. Methylene blue administration in septic shock: a clinical trial. Crit Care Med. 1995; 23(2): 259-64. Weingartner R, Oliveira E, Oliveira ES, Sant'Anna UL, Oliveira RPD, Azambuja LA, Friedman G. Blockade of the action of nitric oxide in human septic shock increases systemic vascular resistance and has detrimental effects on pulmonary function after a short infusion of methylene blue. Braz J Med Biol Res. 1999; doi: https://doi.org/10.1590/S0100-879X1999001200009 Alda M, McKinnon M, Blagdon R, Garnham J, MacLellan S, O'Donovan C,...MacQueen G. Methylene blue treatment for residual symptoms of bipolar disorder: randomized crossover study . Br J Psychiatry. 2017; doi: 10.1192/bjp.bp.115.173930 Farrokhi MR, Lotfi M, Masoudi MS, Gholami M. Effects of methylene blue on postoperative low-back pain and functional outcomes after lumbar open discectomy: a triple-blind, randomized placebo-controlled trial. J Neurosurg Spine. 2016; doi: https://doi.org/10.3171/2015.3.SPINE141172 Sari-Yavuz S, Heck-Swain KL, Keller M, Magunia H, Feng YS, Haeberle HA, ...Koeppen, M. Methylene blue dosing strategies in critically ill adults with shock-A retrospective cohort study. Front Med. 2022; doi: https://doi.org/10.3389/fmed.2022.1014276 Kirov MY, Evgenov OV, Evgenov NV, Egorina EM, Sovershaev MA, Sveinbjørnsson B,...Bjertnaes LJ. Infusion of methylene blue in human septic shock: a pilot, randomized, controlled study . Crit Care Med. 2001; 29(10):1860-7. Memis D, Karamanlioglu B, Yuksel M, Gemlik I, Pamukcu Z. The influence of methylene blue infusion on cytokine levels during severe sepsis . Anaesth Int Care. 2002; doi: https://doi.org/10.1177/0310057X0203000606 Gonçalves-Ferri WA, Albuquerque AAS, de Castro RSAP, Ferreira CHF, Oharomari Jr LK, Silva Lessa DF, Evora PRB. Methylene Blue to Neonatal Septic Shock treatment in neonate pigs . Clinics (Sao Paulo). 2022; doi: https://doi.org/10.1016/j.clinsp.2022.100139 Sandvik L, Erikssen J, Mowinckel P, Roedland EA. A method for determining the size of internal pilot studies. Stat Med. 1996; doi: https://doi.org/10.1002/(SICI)1097-0258(19960730)15:14%3C1587::AID-SIM279%3E3.0.CO;2-F Evora PRB. Methylene blue does not have to be considered only as rescue therapy for distributive shock . J Med Toxicol. 2013; doi: 10.1007/s13181-013-0333-8 Busse LW, Nicholson G, Nordyke RJ, Lee CH, Zeng F, Albertson TE. Angiotensin II for the treatment of distributive shock in the intensive care unit: A US cost-effectiveness analysis. Int J Technol Assess Health Care. 2020; doi: 10.1017/S0266462320000082 van Loo G, Bertrand MJM. Death by TNF: a road to inflammation. Nat Rev Immunol. 2023; doi: https://doi.org/10.1038/s41577-022-00792-3 Wieruszewski PM, Khanna AK. Vasopressor Choice and Timing in Vasodilatory Shock. Crit Care. 2022; doi: https://doi.org/10.1186/s13054-022-03911-7 Venkatesh B, Khanna AK, Cohen J. Less is more: catecholamine-sparing strategies in septic shock. Intensive Care Med. 2019; doi: doi.org/10.1007/s00134-019-05770-3 Kotani Y, Di Gioia A, Landoni G, Belletti A, Khanna AK. An updated "norepinephrine equivalent" score in intensive care as a marker of shock severity . Crit Care. 2023; doi: https://doi.org/10.1186/s13054-023-04322-y Levine AB, Punihaole D, Levine TB. Characterization of the role of nitric oxide and its clinical applications. Cardiology. 2012, doi: https://doi.org/10.1159/000338150 Brennan K, Zheng, F. C XCR2 chemokine receptor. Elsevier Inc; 2007. p. 1-8. de Barros CM, Mello AA, Allodi S. Norepinephrine depresses the nitric oxide production in the ascidian hemocytes. J Invertebr Pathol. 2012; https://doi.org/10.1016/j.jip.2012.07.002 Yamamoto K, Ikeda U, Okada K, Saito T, Shimada K. Arginine vasopressin inhibits nitric oxide synthesis in cytokine-stimulated vascular smooth muscle cells. Hypertens Res. 1997; doi: https://doi.org/10.1291/hypres.20.209 Evora PRB. G-Proteins Agonists and NO/cGMP Blockers: Unexplored Frontiers in the Pharmaceutical Industry. Arq Bras Cardiol. 2017; doi: https://doi.org/10.5935/abc.20170139 Cattaruzza M, Słodowski W, Stojakovic M, Krzesz R, Hecker M. Interleukin-10 induction of nitric-oxide synthase expression attenuates CD40-mediated interleukin-12 synthesis in human endothelial cells. J Biol Chem. 2003; doi: https://doi.org/10.1074/jbc.M301670200 Freels JL, Nelson DK, Hoyt JC, Habib M, Numanami H, Lantz RC, Robbins RA. Enhanced activity of human IL-10 after nitration in reducing human IL-1 production by stimulated peripheral blood mononuclear cells . J Immunol. 2002; doi: https://doi.org/10.4049/jimmunol.169.8.4568 Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.HemodynamicMonitoringinMBandControl.docx SupplementaryTable2.ImmunologicalFeaturesinMBandControl.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3971422","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":274970118,"identity":"b4df0ba7-bbee-4675-b36f-ddb92d2b7404","order_by":0,"name":"Fabio Luis-Silva","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Fabio","middleName":"","lastName":"Luis-Silva","suffix":""},{"id":274970119,"identity":"6ba62f1f-9f59-4f5c-80f0-46aa97aece7a","order_by":1,"name":"Mayra Gonçalves Menegueti","email":"","orcid":"","institution":"University of São 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this condition is associated with an intense systemic inflammatory reaction and is the main cause of morbidity and mortality in patients admitted to intensive care units (ICUs) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], while mortality ranges between 38 and 46.5% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]; however, epidemiological data are scarce and practically nonexistent in developing countries, and a provisional extrapolation of data from high-income countries suggests global estimates of 5.3\u0026nbsp;million deaths annually [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which are serious public health problems.\u003c/p\u003e \u003cp\u003eA diagnosis of septic shock occurs in patients with a confirmed or presumed focus of infection associated with a mean arterial pressure (MAP)\u0026thinsp;\u0026le;\u0026thinsp;65 mmHg and a lactate concentration\u0026thinsp;\u0026ge;\u0026thinsp;2.0 mmol/L after adequate volume replacement [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTreatment includes fluid replacement, infusion of vasopressors, and administration of antibiotics within the first hour [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]; these treatments are associated with low-dose corticosteroids in refractory cases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], but mortality remains high, especially in developing countries. Studying new medications that help maintain hemodynamic stability until the antibiotic acts and combats the infectious focus is necessary [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMethylene blue (MB), a nonselective inhibitor of soluble guanylate cyclase (SGC) and nitric oxide synthase (NOS), is a heterocyclic aromatic compound from the phenothiazine class that has been used since the 19th century and has had proven hemodynamic effects since 1976 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]; additionally, MB has been used safely, with few side effects when it is used at adequate doses. The safety of intravenous administration and dosages of 1 to 3 mg/kg have been previously reported, and the authors state that excessive doses of MB result in adverse effects on visceral tissue perfusion. Doses greater than 40 mg/kg are lethal [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral studies have proposed that MB can treat circulatory shock secondary to vasoplegia via nitric oxide (NO) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In a rat model of endotoxemia induced by lipopolysaccharide (LPS) and treated with MB, complete prevention of leukocyte rolling and adhesion to the endothelium was observed, resulting in a consistent systemic blood pressure response and a decreased need for vasoactive medications [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In animal endotoxemia and in patients with septic shock, decreasing plasma levels of stable nitric oxide (NO) metabolites, nitrites, nitrates, and cGMP were demonstrated [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although the medical literature does not include robust studies on the use of MB in septic shock, even with a small sample, the results are promising regarding the association of MB with conventional treatment in these patients [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In theory, the inhibition of excessive NO could act favorably, preventing systemic vasodilation and reducing microvascular injury in septic individuals. Another theory is that MB improves mitochondrial function and adenosine triphosphate (ATP) production in cells, helping to reverse cardiovascular dysfunction associated with septic shock [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome studies have used MB to reverse vasoplegia during the postoperative period after major surgery [\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], during anaphylactic shock, during refractory shock in traumatized patients and during the postoperative period after liver transplantation [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Other studies have shown that reducing vasopressors and maintaining adequate MAP levels are beneficial for patients with septic shock [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], but these are small studies and lack robust evidence for this approach.\u003c/p\u003e \u003cp\u003eA retrospective cohort study [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] also showed the benefit of MB in different types of circulatory shock (septic, cardiogenic and vasoplegic), with a reduced need for noradrenaline and reduced mortality after 28 days of observation; however, these findings highlight the importance of performing a prospective and randomized study.\u003c/p\u003e \u003cp\u003eA randomized clinical trial in patients with septic shock showing that the administration of MB within 24 hours reduced the time to vasopressor discontinuation did not evaluate possible hemodynamic changes, inflammatory mediators, or the dosage of nitrite or nitrate [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe use of MB was also described in two randomized clinical trials evaluating its use in the treatment of septic shock at an early stage (less than 24 hours after diagnosis), which demonstrated a reduction in and early weaning of vasopressors [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, these studies did not use invasive hemodynamic monitoring or the measurement of inflammatory or anti-inflammatory mediators or NO for 72 hours, as in the present study.\u003c/p\u003e \u003cp\u003eStudies have shown the beneficial effects of MB in patients with septic shock [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]; however, studies involving patients in the acute phase are rare [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur hypothesis is that MB contributes to reducing the infusion of vasopressors, improving tissue perfusion and delaying mitochondrial death induced by nitric oxide if administered in the first 48 hours of septic shock. Therefore, the aim of the present study was to determine whether the administration of MB to patients in the initial phase of septic shock leads to a reduction in the use of vasopressors compared to that in the control group.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design, population and sampling\u003c/h2\u003e \u003cp\u003eThis was a pilot randomized clinical trial conducted in the ICU of a tertiary university hospital from January 2019 to August 2023. All the legal guardians of the included patients agreed and signed the free and informed consent form. The Ethics Committee of the Hospital das Cl\u0026iacute;nicas of the Faculty of Medicine of Ribeir\u0026atilde;o Preto approved this protocol\u0026ndash;number: 562/2017, version: 2/2016.Blinding was not possible since MB leaves body fluids with a bluish-green color are easily identified upon use. To minimize possible biases, the professional responsible for randomization had no access to patient clinical records, and the researcher did not know the patient group when performing hemodynamic monitoring for data collection.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the study population and study design. Eighty patients were enrolled upon signing the informed consent form by their next of kin and were randomly allocated into two groups according to the previously published protocol [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. After withdrawing based on clinical status (n\u0026thinsp;=\u0026thinsp;48), a total of 42 patients were included in the present study; 23 patients were allocated to the control group, and 19 were randomized to the MB group. No previous studies in the literature have used continuous-dose MB for 48 hours to support the sample size calculation. Therefore, in the present investigation, the sample size was defined according to the guidelines proposed by Sandvik et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] for pilot clinical trials, which recommended the inclusion of 20 patients in each study arm. After measuring and collecting initial exams, patients in the MB group received conventional treatment and MB at a dose of 3 mg/kg in 20 min and then 0.5 mg/kg/h for 48 hours, and patients in the control group received conventional treatment. Conventional treatment included fluid replacement, vasopressor drugs (noradrenaline and vasopressin), hydrocortisone (200 mg/day) and antibiotics within the first hour. After completion of the protocol, patients were followed up for 30 days to assess the outcome, discharge status, or death status. After randomization, the following procedures were performed: weight measurement using a bed scale (Styker\u0026reg; brand), catheter insertion in the femoral vein (VolumeView System\u0026reg;), and central venous access in the internal jugular or subclavian vein (Presep\u0026reg;) for monitoring with the EV1000\u0026reg; platform (Edwards Lifesciences Corporation\u0026reg;). The SAPS3 (Simplified Acute Physiology Score 3) and APACHE II (Acute Physiology and Chronic Health Evaluation) prognostic scores were calculated for the first 24 hours after admission, and the daily SOFA score was calculated for the four days of the protocol. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the demographic and clinical characteristics of the study population. Considering the patients with positive cultures, empirical antibiotics were started less than three hours after the diagnosis of septic shock, providing adequate coverage in 43.5% of the control group and 21.1% of the MB group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.1240).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic and clinical features of the study population.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;23)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMB (n\u0026thinsp;=\u0026thinsp;19)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep value*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSex (Male) \u0026ndash; n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14 (74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.7700\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge (Years) \u0026ndash; Mean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.7\u0026thinsp;\u0026plusmn;\u0026thinsp;17.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51.5\u0026thinsp;\u0026plusmn;\u0026thinsp;14.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0876\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHypertension \u0026ndash; n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (32)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.9300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDiabetes \u0026ndash; n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.8900\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAcute kidney injury \u0026ndash; n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHemodialysis \u0026ndash; (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSAPS3 \u0026ndash; Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58.2\u0026thinsp;\u0026plusmn;\u0026thinsp;9.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e78.1\u0026thinsp;\u0026plusmn;\u0026thinsp;11.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.0001\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDeath Risk \u0026ndash; Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.1\u0026thinsp;\u0026plusmn;\u0026thinsp;20.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82.0\u0026thinsp;\u0026plusmn;\u0026thinsp;13.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.0001\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAPACHE II \u0026ndash; Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026lt;\u0026thinsp;0.0001\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSOFA \u0026ndash; Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT1 (Baseline)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0.0010\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT4 (24 hours)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0.0040\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT5 (48 hours)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0.0020\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eT6 (72 hours)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0.0040\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSites of infection\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAbdomen \u0026ndash; n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (21.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (36.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLung \u0026ndash; n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15 (65.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (31.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0.0300\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUrinary tract \u0026ndash; n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (4.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (15.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2450\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCRBSI \u0026ndash; n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (4.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3280\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOther \u0026ndash; n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (4.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (15.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2450\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePositive culture \u0026ndash; n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (56.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (68.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.4390\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEffectiveness of empiric antibiotic \u0026ndash; (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1240\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDays of antibiotic use \u0026ndash; Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.9\u0026thinsp;\u0026plusmn;\u0026thinsp;20.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.4\u0026thinsp;\u0026plusmn;\u0026thinsp;12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.6386\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eShock time at baseline (hour) \u0026ndash; Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.5\u0026thinsp;\u0026plusmn;\u0026thinsp;24.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.3\u0026thinsp;\u0026plusmn;\u0026thinsp;12.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0738\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFluid balance (mL in 72 hours) \u0026ndash; Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,416\u0026thinsp;\u0026plusmn;\u0026thinsp;3,158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,871\u0026thinsp;\u0026plusmn;\u0026thinsp;3,163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1453\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e30 days outcome (Death) \u0026ndash; n (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9 (47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.3800\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e* MB: Methylene blue; SD: standard deviation; CRBSI \u0026ndash; Catheter-Related Blood Stream Infection. Chi-square test was used for categorical data and Student\u0026rsquo;s test employed for continuous variables comparisons. Significant differences are underscored in italic.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHemodynamic and Vasopressor Drug Monitoring\u003c/h2\u003e \u003cp\u003eAfter calibrating the EV1000\u0026reg; platform, the mean arterial pressure (MAP), cardiac output (CO), cardiac index (CI), heart rate (HR), systemic vascular resistance index (SVRI), central venous pressure (CVP), stroke volume index (SVI), stroke volume variation (SVV), global ejection fraction (GEF), pulmonary vascular permeability index (PVPI), global end diastolic volume index (GEDVI) and extravascular lung water index (ELWI) were measured. Measurements of central venous oxygen saturation (ScVO\u003csub\u003e2\u003c/sub\u003e), arterial blood gas, serum lactate, bilirubin, complete blood count, sodium, potassium, urea, and creatinine were also performed. The oxygen delivery index (DO\u003csub\u003e2\u003c/sub\u003eI), oxygen consumption index (VO\u003csub\u003e2\u003c/sub\u003eI), and oxygen extraction rate (O\u003csub\u003e2\u003c/sub\u003eER) were calculated. Vasopressor drug monitoring (norepinephrine/NOR and vasopressin/VAS) was carried out daily by a physician according to a standardized institutional protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLaboratory analysis of immunological features\u003c/h2\u003e \u003cp\u003eTo evaluate the immunological parameters, serial whole-blood sampling was performed along the study timeline (T1 to T6) using EDTA and heparin. Samples were subjected to centrifugation at 3,500 rpm for 10 minutes at 16\u0026deg;C, and plasma aliquots were stored at -80\u0026deg;C until processing. Soluble immune mediator (interleukin 8 [IL-8/CXCL8], interleukin 6 [IL-6], tumor necrosis factor alpha [TNF-α], and interleukin 10 [IL-10]) levels were measured in EDTA-treated samples according to the manufacturer\u0026rsquo;s instructions (R\u0026amp;D Systems), and nitric oxide (NO\u003csub\u003e3\u003c/sub\u003e) levels were measured in heparin-treated samples via chemiluminescence according to the manufacturer\u0026rsquo;s instructions (Sievers NO Analizer).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe chi-square test was used to verify the associations of qualitative variables between the MB and control groups. Student\u0026rsquo;s t test was used to analyze quantitative clinical variables. The comparison of immunological features between the MB and control groups at each timepoint was performed by the Mann‒Whitney test. The Spearman rank test was used to perform cross-correlation analysis of vasopressor drugs and hemodynamic and immunological variables (attributes) among all timepoints. GraphPad Prism software was used for all the statistical analyses. In all cases, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance. Significant correlations were used to construct integrative networks. Integrative networks were built using the systems biology approach of the Cytoscape open-source platform (available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cytoscape.org\u003c/span\u003e\u003cspan address=\"https://cytoscape.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) based on the \u0026ldquo;r\u0026rdquo; scores of significant correlations. The networks were assembled using a cluster layout with nodes used to represent each variable, and connecting lines were employed to identify positive (continuous line) and negative (dashed line) correlations. The node sizes are proportional to the number of correlations between parameters. Line thickness illustrates the correlation strength, ranging from weak/moderate (\u0026ldquo;r\u0026rdquo; scores from |0.1 to 0.6|, thin lines) to strong correlations (\u0026ldquo;r\u0026rdquo; scores from \u0026ge; |0.7|, thick lines). The red line illustrates the correlations between immunological features and vasopressor drugs. Correlation matrices were assembled using the \u0026ldquo;corrplot\u0026rdquo; package of R software (Project for Statistical Computing Version 3.0.1). Microsoft Excel and Prism GraphPad software were used to create the graphics.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMAP and vasopressor doses along the kinetic timeline in the MB and control groups\u003c/h2\u003e \u003cp\u003eThe analysis of MAP and vasopressor doses along the kinetic timeline was assessed by comparing pairs of adjacent timepoints. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the median MAP values along with the NOR and VAS scores along the kinetic timelines in the MB and control groups. The data analysis did not reveal significant differences in the MAP during the kinetics follow-up. The NOR dose was markedly lower in the MB test at T2 than at T1, with a continuous decrease toward T5, while in the control group, a decrease in NOR was observed later at T4 than at T3. A significant difference in NOR dose between the MB and control groups was observed at T3. The analysis of VAS scores demonstrated early withdrawal in the MB group at T4, while in the control group, VAS scores were withdrawn later at T5. A significant difference in VAS score between the MB and control groups was observed at T4. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Analysis of several hemodynamic variables (CO, CI, HR, SVRI, CVP, SVI, SVV, GEF, PVPI, GEDVI and ELWI) along the kinetics timeline (T1 to T6) did not reveal significant differences (data not shown). A detailed description of the median values of MAP and vasopressor doses along the kinetic timeline is provided in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cem\u003eKinetic timeline of serum lactate concentration, DO\u003c/em\u003e \u003csub\u003e \u003cem\u003e2\u003c/em\u003e \u003c/sub\u003e \u003cem\u003eI, VO\u003c/em\u003e \u003csub\u003e \u003cem\u003e2\u003c/em\u003e \u003c/sub\u003e \u003cem\u003eI and O\u003c/em\u003e \u003csub\u003e \u003cem\u003e2\u003c/em\u003e \u003c/sub\u003e \u003cem\u003eER \u0026times; CO in the MB and control groups\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe analysis of hemodynamic variables (serum lactate concentration, DO\u003csub\u003e2\u003c/sub\u003eI, VO\u003csub\u003e2\u003c/sub\u003eI and O\u003csub\u003e2\u003c/sub\u003eER \u0026times; CO) was assessed along the kinetic timeline by comparing pairs of adjacent timepoints. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the median values of the serum lactate concentration, DO\u003csub\u003e2\u003c/sub\u003eI, VO\u003csub\u003e2\u003c/sub\u003eI and O\u003csub\u003e2\u003c/sub\u003eER \u0026times; CO. Data analysis demonstrated that while the serum lactate concentration decreased in the MB group at T2 compared to T1, no differences were observed between adjacent timepoints in the control group. The analysis of DO\u003csub\u003e2\u003c/sub\u003eI demonstrated an early increase in MB at T2 and T3 and a later increase at T5 in the control group, leading to differences between the MB and control groups. No significant differences were observed for VO\u003csub\u003e2\u003c/sub\u003eI or O\u003csub\u003e2\u003c/sub\u003eER \u0026times; CO along the kinetics timeline. The analysis of fold changes further corroborated these findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A detailed description of the median values of the serum lactate concentration, DO\u003csub\u003e2\u003c/sub\u003eI, VO\u003csub\u003e2\u003c/sub\u003eI and O\u003csub\u003e2\u003c/sub\u003eER \u0026times; CO concentration along the kinetic timeline is provided in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eChanges in plasma immune mediator and nitric oxide levels in the MB and control groups\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e presents the overall profile of soluble plasma immune mediators and NO in the MB and control groups. These immune mediators were chosen because they are related to the inflammatory response in sepsis, thus making it possible to evaluate whether treatment with MB alters the inflammatory response. The analysis of immune mediators and NO was assessed along the kinetic timeline by comparing pairs of adjacent timepoints. The data analysis demonstrated an increase in CXCL8 in the MBs at T6 compared to T5 and a decrease in the control group at T5 compared to T4. The data showed an increase in IL-6 in the control group at T2, with a progressive decrease toward T5 and a decrease in MB at T4 compared to T3. Compared with those in the control group, TNF-α in the MB at T5 were lower than those in T4, and lower levels were detected in the MB at T5 and T6. The analysis of IL-10 showed lower levels in the control group at T2 than at T1. Higher levels of IL-10 were observed in the MBs at T1 and T6 than in the control group. NO was elevated at T3 in the control group and progressively decreased toward T5. In the MB group, the levels of NO were greater than those in the control group at T4 and T5 and displayed a progressive decrease toward T6. The analysis of fold changes corroborated these findings. A detailed description of the median values of plasma immune mediators and nitric oxide concentrations along the kinetic timeline is provided in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIntegrative networks of vasopressor drugs and hemodynamic and immunological variables in the MB and control groups\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e displays the integrative network of vasopressor drugs and hemodynamic and immunological variables in the MB and control groups. Integrative networks were built using systems biology approaches with a cluster layout with nodes used to represent each variable and connecting lines used to identify positive and negative correlations between pairs of attributes. The node sizes are proportional to the number of correlations. Despite the similar numbers of correlations observed in the MB and control groups (n\u0026thinsp;=\u0026thinsp;48 and n\u0026thinsp;=\u0026thinsp;49, respectively), intracluster analysis demonstrated greater contributions of the \u0026ldquo;immune mediators\u0026rdquo; (32% \u003cem\u003evs\u003c/em\u003e 28%) and \u0026ldquo;MAP;NOR;VAS\u0026rdquo; clusters in the MB (19% \u003cem\u003evs\u003c/em\u003e 16%) cohort than in the control group. Conversely, more correlations within the \u0026ldquo;Hemodynamic Monitoring\u0026rdquo; (56% \u003cem\u003evs\u003c/em\u003e 49%) cluster were observed in the Control group than in the MB group. Notably, while NOR and VAS scores were directly correlated with NO in the control group, they were directly correlated with IL-10 in the MB group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, red connecting lines). Overall, the integrative network analysis demonstrated that, while NO represents a key attribute orchestrating the correlation with the \u0026ldquo;Hemodynamic Monitoring\u0026rdquo; cluster in the control group, IL-10 plays a pivotal role in coordinating the correlation with the \u0026ldquo;Hemodynamic Monitoring\u0026rdquo; cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, thick connecting lines). This was more evident for the ScVO\u003csub\u003e2\u003c/sub\u003e, O\u003csub\u003e2\u003c/sub\u003eER and O\u003csub\u003e2\u003c/sub\u003eER \u0026times; CO attributes. Taken together, these findings indicate that the underlying mechanism through which MBs impact hemodynamic features in septic shock may include not only the suppression of NO activity but also the involvement of other events mediated by IL-10. Detailed correlogram data supporting the integrative network are provided in Supplementary Fig.\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study used MB to reduce NO-mediated vasoplegia, which can minimize the use of vasopressors, in addition to being a low-cost medication, without side effects and being easily accessible in health units [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe started MB in the first 72 hours of septic shock and observed a reduction in the dose of NOR immediately after the start of the infusion compared to that in the control group, where this reduction occurred only after 24 hours. Similar findings were described in a randomized clinical trial that demonstrated that early use of MB reduced the duration of vasopressor use compared to that in the control group, and most importantly, no serious adverse effects were detected. The authors further suggest that MB should not be used as rescue therapy but rather as adjuvant therapy in the early stages of septic shock. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Due to its safety profile, greater availability and lower cost than other catecholamine-sparing agents [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], MB could emerge as a viable therapy within a multimodal strategy to maintain MAP and improve tissue perfusion. Furthermore, MB contributes to reducing the use of high-dose vasopressors [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], which was also found in our study.\u003c/p\u003e \u003cp\u003eWhen evaluating weaning from VAS, we observed early withdrawal in the MB group within 24 hours, while in the control group, VAS withdrawal was observed within 48 hours.\u003c/p\u003e \u003cp\u003eThe study of Preiser et al.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] investigated the intravenous administration of MB at a single dose of 2 mg/kg in patients with septic shock and demonstrated an increase in MAP and IRVS, but these hemodynamic improvements were not sustained, suggesting that longer MB administration deserves further investigation. In our study, we performed a continuous infusion of MB for 48 hours and observed a decrease in the use of vasopressors within 2 hours that was maintained during the period of MB infusion. Perhaps this time is still short since many patients in the present study still needed the use of vasopressors, so the shock had not yet been fully resolved. Furthermore, there was a greater need for vasopressors after MB suspension. We speculate that in septic shock, a longer time is needed for complete hemodynamic restoration since the effectiveness of the antimicrobial agent is important and is linked to access to the culture results.\u003c/p\u003e \u003cp\u003eA randomized clinical trial in patients with septic shock showed that the administration of MB within 24 hours reduced the duration of vasopressor suspension but did not evaluate possible hemodynamic changes, inflammatory mediators, nitrites or nit dosages [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, we observed that the dosage of nitrate (a NO metabolite) in the MB group was greater than that in the control group during the first 48 hours (T4). This can be explained by the fact that MB, by blocking guanylate cyclase, increases NO, but it remains inactive because it depends on this enzyme. This high dosage of nitrate in the MB group may have been beneficial since NO deficiency can lead to endothelial dysfunction, increased insulin resistance and impairment of the immune system [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother important aspect of MB is its antioxidant properties, which eliminate reactive oxygen species (ROS) independently of cGMP, helping to protect cells against oxidative damage or even helping to improve mitochondrial function [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, few robust studies have evaluated the dosage of cytokines in patients who use MB for septic shock. Memis et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] evaluated the dosage of TNF-α, IL-1, IL-2, IL-6 and CXCL8 and found no changes in serum levels during 6 hours of MB use. We speculate that these results differ from our study, as prolonged use of MB could trigger more sustained effects on inflammatory and anti-inflammatory responses.\u003c/p\u003e \u003cp\u003eIn the present study, we also observed an increase in CXCL8 in the MB group at T2 to T3, which may have contributed to improved targeting of granulocytes, benefiting the acute phase of septic shock [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the control group, there was a progressive reduction from T1 to T5; however, there was an increase from T4 to T6, at which point we already expected a reduction due to the control of the inflammatory and infectious process.\u003c/p\u003e \u003cp\u003eIn the control group, the TNF-α concentration progressively increased, mainly after T3, and remained elevated until T6, which may indicate an excessive response to the infection, even 72 hours after the start of treatment. In the MB group, there was stabilization between T1 and T2, with a decrease in T3 and a much less pronounced increase in T4, with a subsequent decrease until T5, which increased again after withdrawal of the maintenance dose of MB in T6. Although patients in the MB group had more severe illnesses according to prognostic indices, we noticed that in the MB group, there was a lower increase in the level of this proinflammatory cytokine. TNF-α plays an important role in septic shock, signaling the path that defense cells must follow, activating the immune system and increasing the antigen-specific response; however, excessive TNF-α activation is harmful, leading to cell death [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIL-10, an anti-inflammatory cytokine, was expressed at lower levels from T1 to T4 in the control group than in the MB group, with increases in T5 and T6 in the MB group compared to the control group. This elevation observed in the MB group may play a role in maintaining homeostasis between inflammatory and anti-inflammatory mediators.\u003c/p\u003e \u003cp\u003eIn this study, we also analyzed the integrative networks of vasopressor drugs and hemodynamic and immunological variables in the MB and control groups. The data analysis demonstrated that while NOR and VAS scores were directly correlated with NO in the control group, they were directly correlated with IL-10 in the MB group. These findings demonstrate that the mechanisms underlying the effect of MB on hemodynamic characteristics in septic shock may rely not only on blocking the action of NO but also on other events mediated by IL-10, which may be one of the possibilities for hemodynamic improvement, independent of action and serum nitrate levels.\u003c/p\u003e \u003cp\u003eIn addition to the vasoconstrictor effect, norepinephrine and vasopressin can also act by decreasing the production of nitric oxide [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Thus, in the control group, in which MB was not used, vasopressors showed a direct and strong correlation with nitrate. However, when we used MBs, there was no correlation between the use of vasopressors and nitrate. As mentioned above, when guanylate cyclase is blocked, NO levels remain elevated but are inactive. Furthermore, guanylate cyclase, when inactivated, prevents the conversion of GTP into cGMP, which, in addition to reducing vasodilation, also facilitates the action of noradrenaline via cAMP since the cGMP pathway, when little activated, releases cAMP [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Furthermore, in the MB group, vasopressors showed a strong and direct correlation with IL-10, which also affects nitrate reduction [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFinally, we also observed that patients in the MB group had more severe disease according to the SOFA, SAPS3 and APACHE II scores. However, patients in the MB group used fewer days of vasopressors and had a mortality rate of 47.37% versus 60.87% in the control group. This lower mortality may be related to a balance between inflammatory and anti-inflammatory mediators, which is associated with the theory that describes an improvement in mitochondrial function and the production of adenosine triphosphate (ATP) in cells, helping to reverse cardiovascular dysfunction associated with septic shock [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome limitations must be recognized. First, this was a single-center study; second, it was an unblinded study, as highlighted in the methodology section.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eEarly methylene blue (MB) administration alongside standard septic shock treatment reduces vasopressor doses, possibly involving nitric oxide (NO) mechanisms. A possible mechanism of action may involve modulation of inflammatory and anti-inflammatory mediators, enhancing immune response. However, larger and longer studies are needed for validation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAPACHE II = Acute Physiology\u0026nbsp;and\u0026nbsp;Chronic Health Evaluation;\u0026nbsp;ATP = Adenosina Triphosphate;\u0026nbsp;MB = Methylene Blue;\u0026nbsp;CI = Cardiac Index;\u0026nbsp;cNOS = Constitutive Nitric Oxide Synthase;\u0026nbsp;CO = Cardiac Output;\u0026nbsp;SGC = Soluble Guanylate Cyclase;\u0026nbsp;DO\u003csub\u003e2\u003c/sub\u003e =\u0026nbsp;Oxygen Delivery;\u0026nbsp;GEF = Global Ejection Fraction;\u0026nbsp;HR = Heart Rate;\u0026nbsp;PEWI = Pulmonary Extravascular Water Index;\u0026nbsp;ICU = Intensive Care Unit;\u0026nbsp;GEDVI = Global End-Diastolic Volume Index;\u0026nbsp;iNOS = Induced Nitric Oxide Synthase;\u0026nbsp;PVPI = Pulmonary Vascular Permeability Index;\u0026nbsp;SVRI = Systemic Vascular Resistance Index;\u0026nbsp;SVI = Systolic Volume Index;\u0026nbsp;MAP = Mean Arterial Pressure;\u0026nbsp;NO = Nitric Oxide;\u0026nbsp;NOS = Nitric Oxide Synthase;\u0026nbsp;CVP = Central Venous Pressure;\u0026nbsp;SAPS3 = Simplified Acute Physiology Score;\u0026nbsp;SOFA = Sequential Organ Failure Assessment;\u0026nbsp;ScVO\u003csub\u003e2\u003c/sub\u003e = Central Venous Oxygen Saturation;\u0026nbsp;SVV = Systolic Volume Variation;\u0026nbsp;VO\u003csub\u003e2\u003c/sub\u003e: Oxygen Consumption.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank everyone involved in the implementation of this project, especially the medical and nursing staff of the Hospital das Cl\u0026iacute;nicas Ribeir\u0026atilde;o Preto University of S\u0026atilde;o Paulo.\u0026nbsp;MAM\u0026nbsp;thank\u0026nbsp;the\u0026nbsp;Funda\u0026ccedil;\u0026atilde;o de Apoio ao Ensino, Pesquisa e Assist\u0026ecirc;ncia (FAEPA) of\u0026nbsp;the\u0026nbsp;Clinics Hospital of Ribeir\u0026atilde;o Preto and FAPESP for the grant (#2018/26553-0) and the research fellowships program (#2021/01195-6). OAMF received PQ fellowships from CNPq. OAMF is\u0026nbsp;a\u0026nbsp;research fellow from\u0026nbsp;the\u0026nbsp;Universidade do Estado do Amazonas-UEA (PROVISIT No. 005/2023-PROPESP/UEA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFLS and MAM wrote the paper; FLS, MGM, LMP, CRS, BCPM, MDD, BLM, FBS and GHMR were responsible for data collection for the study; MAM, OAMF, JPBS and IACR prepared tables and figures; MAM, MGM and OAMF were responsible for interpretation and statistical analysis; MCJ, FM and CB were responsible to laboratorial analysis and MAM, MGM, PRBE and ABF reviewed the manuscript. Individual patient data will remain confidential.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe received funding from FAPESP through the grant (#2018/26553-0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analysis were carried out using GraphPad Prism software, version8.0, (San Diego, CA, USA). All participants received a numerical identification from the study itself. Personal data will be stored in the hospital\u0026rsquo;s digital system, password protected to ensure confidentiality. Any modifications or adverse events related to the protocol were communicated to REBEC.gov and to the Research Ethics Committee and Clinical Research Unit of Hospital das Cl\u0026iacute;nicas da Faculdade de Medicina de Ribeir\u0026atilde;o Preto by the researcher in charge. All principal investigators have access to the final trial dataset. Datasets used and/ or analyzed during the current study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis clinical trial is registered at REBEC\u0026nbsp;(https://ensaiosclinicos.gov.br/rg/RBR-96584w4)\u0026nbsp;on June 12, 2021. The Ethics Committee of the Hospital das Cl\u0026iacute;nicas of the Faculty of Medicine of Ribeir\u0026atilde;o Preto approved this protocol\u0026ndash;number: 562/2017, version: 2/2016.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTerms of free and informed consent were collected from all study participants through their legal guardians.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJuffermans NP, Vervloet MG, Daemen-Gubbels CR, Binnekade JM, de Jong M, Groeneveld AJ. 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Transfus Apher Sci. 2006; doi: https://doi.org/10.1016/j.transci.2006.08.008\u003c/li\u003e\n\u003cli\u003eRojas JC, Bruchey AK, Gonzalez-Lima F. Neurometabolic mechanisms for memory enhancement and neuroprotection of methylene blue\u003cem\u003e.\u003c/em\u003e Prog Neurobiol. 2012; doi: https://doi.org/10.1016/j.pneurobio.2011.10.007\u003c/li\u003e\n\u003cli\u003eKim JH, Kim JH, Ku NS, Kim YJ, Kim HB, Seok H, Lee DG,... Park DW. Korean Registry for Improving Sepsis Survival (KISS): Protocol for a Multicenter Cohort of Adult Patients with Sepsis or Septic Shock. Infect Chemot. 2020; doi: https://doi.org/10.3947/ic.2020.52.1.31\u003c/li\u003e\n\u003cli\u003ePaciullo CA, Methylene blue for the treatment of septic shock\u003cem\u003e.\u003c/em\u003e Pharmacotherapy, 2010; doi: https://doi.org/10.1592/phco.30.7.702\u003c/li\u003e\n\u003cli\u003eAnnane D, Annane D, Aegerter P, Jars-Guincestre MC, Guidet B. Current epidemiology of septic shock: the CUB-R\u0026eacute;a Network\u003cem\u003e.\u003c/em\u003e Am J Respir Crit Care Med. 2003; doi: https://doi.org/10.1164/rccm.2201087\u003c/li\u003e\n\u003cli\u003eSchneider F, Lutun PH, Hasselmann M, Stoclet JC, Tempe JD. Methylene blue increases systemic vascular resistance in human septic shock. Preliminary observations. IntCare Med. 1992; doi: https://doi.org/10.1007/BF01706481\u003c/li\u003e\n\u003cli\u003eRivers E, Nguyen B, Havstad S, Ressler J, Muzzin A, Knoblich B,...Tomlanovich M. Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med. 2001; doi: 10.1056/NEJMoa010307\u003c/li\u003e\n\u003cli\u003eDaemen-Gubbels CR, Groeneveld PH, Groeneveld AJ, van Kamp GJ, Bronsveld W, Thijs LG. Methylene blue increases myocardial function in septic shock. Crit Care Med. 1995; 23(8):1363-70.\u003c/li\u003e\n\u003cli\u003eGachot B, Bedos, JP, Veber B, Wolff M, Regnier B. Short-term effects of methylene blue on hemodynamics and gas exchange in humans with septic shock. Intensive Care Med. 1995; doi: https://doi.org/10.1007/BF01700666\u003c/li\u003e\n\u003cli\u003ePreiser JC, Lejeune P, Roman A, Carlier E, De Backer D, Leeman M,...Vincent JL. Methylene blue administration in septic shock: a clinical trial. Crit Care Med. 1995; 23(2): 259-64.\u003c/li\u003e\n\u003cli\u003eWeingartner R, Oliveira E, Oliveira ES, Sant\u0026apos;Anna UL, Oliveira RPD, Azambuja LA, Friedman G. Blockade of the action of nitric oxide in human septic shock increases systemic vascular resistance and has detrimental effects on pulmonary function after a short infusion of methylene blue. Braz J Med Biol Res. 1999; doi: https://doi.org/10.1590/S0100-879X1999001200009\u003c/li\u003e\n\u003cli\u003eAlda M, McKinnon M, Blagdon R, Garnham J, MacLellan S, O\u0026apos;Donovan C,...MacQueen G. Methylene blue treatment for residual symptoms of bipolar disorder: randomized crossover study\u003cem\u003e.\u003c/em\u003e Br J Psychiatry. 2017; doi: 10.1192/bjp.bp.115.173930\u003c/li\u003e\n\u003cli\u003eFarrokhi MR, Lotfi M, Masoudi MS, Gholami M. Effects of methylene blue on postoperative low-back pain and functional outcomes after lumbar open discectomy: a triple-blind, randomized placebo-controlled trial. J Neurosurg Spine. 2016; doi: https://doi.org/10.3171/2015.3.SPINE141172 \u003c/li\u003e\n\u003cli\u003eSari-Yavuz S, Heck-Swain KL, Keller M, Magunia H, Feng YS, Haeberle HA, ...Koeppen, M. Methylene blue dosing strategies in critically ill adults with shock-A retrospective cohort study. Front Med. 2022; doi: https://doi.org/10.3389/fmed.2022.1014276\u003c/li\u003e\n\u003cli\u003eKirov MY, Evgenov OV, Evgenov NV, Egorina EM, Sovershaev MA, Sveinbj\u0026oslash;rnsson B,...Bjertnaes LJ. Infusion of methylene blue in human septic shock: a pilot, randomized, controlled study\u003cem\u003e.\u003c/em\u003e Crit Care Med. 2001; 29(10):1860-7.\u003c/li\u003e\n\u003cli\u003eMemis D, Karamanlioglu B, Yuksel M, Gemlik I, Pamukcu Z. The influence of methylene blue infusion on cytokine levels during severe sepsis\u003cem\u003e.\u003c/em\u003e Anaesth Int Care. 2002; doi: https://doi.org/10.1177/0310057X0203000606\u003c/li\u003e\n\u003cli\u003eGon\u0026ccedil;alves-Ferri WA, Albuquerque AAS, de Castro RSAP, Ferreira CHF, Oharomari Jr LK, Silva Lessa DF, Evora PRB. Methylene Blue to Neonatal Septic Shock treatment in neonate pigs\u003cem\u003e.\u003c/em\u003e Clinics (Sao Paulo). 2022; doi: https://doi.org/10.1016/j.clinsp.2022.100139\u003c/li\u003e\n\u003cli\u003eSandvik L, Erikssen J, Mowinckel P, Roedland EA. A method for determining the size of internal pilot studies. Stat Med. 1996; doi: https://doi.org/10.1002/(SICI)1097-0258(19960730)15:14%3C1587::AID-SIM279%3E3.0.CO;2-F\u003c/li\u003e\n\u003cli\u003eEvora PRB. Methylene blue does not have to be considered only as rescue therapy for distributive shock\u003cem\u003e.\u003c/em\u003e J Med Toxicol. 2013; doi: 10.1007/s13181-013-0333-8\u003c/li\u003e\n\u003cli\u003eBusse LW, Nicholson G, Nordyke RJ, Lee CH, Zeng F, Albertson TE. Angiotensin II for the treatment of distributive shock in the intensive care unit: A US cost-effectiveness analysis. Int J Technol Assess Health Care. 2020; doi: 10.1017/S0266462320000082\u003c/li\u003e\n\u003cli\u003evan Loo G, Bertrand MJM. Death by TNF: a road to inflammation. Nat Rev Immunol. 2023; doi: https://doi.org/10.1038/s41577-022-00792-3\u003c/li\u003e\n\u003cli\u003eWieruszewski PM, Khanna AK. Vasopressor Choice and Timing in Vasodilatory Shock. Crit Care. 2022; doi: https://doi.org/10.1186/s13054-022-03911-7\u003c/li\u003e\n\u003cli\u003eVenkatesh B, Khanna AK, Cohen J. Less is more: catecholamine-sparing strategies in septic shock. Intensive Care Med. 2019; doi: doi.org/10.1007/s00134-019-05770-3\u003c/li\u003e\n\u003cli\u003eKotani Y, Di Gioia A, Landoni G, Belletti A, Khanna AK. An updated \u0026quot;norepinephrine equivalent\u0026quot; score in intensive care as a marker of shock severity\u003cem\u003e.\u003c/em\u003e Crit Care. 2023; doi: https://doi.org/10.1186/s13054-023-04322-y\u003c/li\u003e\n\u003cli\u003eLevine AB, Punihaole D, Levine TB. Characterization of the role of nitric oxide and its clinical applications. Cardiology. 2012, doi: https://doi.org/10.1159/000338150\u003c/li\u003e\n\u003cli\u003eBrennan K, Zheng, F. C\u003csub\u003eXCR2\u003cem\u003e \u003c/em\u003e\u003c/sub\u003echemokine receptor. Elsevier Inc; 2007. p. 1-8.\u003c/li\u003e\n\u003cli\u003ede Barros CM, Mello AA, Allodi S. Norepinephrine depresses the nitric oxide production in the ascidian hemocytes. J Invertebr Pathol. 2012; https://doi.org/10.1016/j.jip.2012.07.002\u003c/li\u003e\n\u003cli\u003eYamamoto K, Ikeda U, Okada K, Saito T, Shimada K. Arginine vasopressin inhibits nitric oxide synthesis in cytokine-stimulated vascular smooth muscle cells. Hypertens Res. 1997; doi: https://doi.org/10.1291/hypres.20.209\u003c/li\u003e\n\u003cli\u003eEvora PRB. G-Proteins Agonists and NO/cGMP Blockers: Unexplored Frontiers in the Pharmaceutical Industry. Arq Bras Cardiol. 2017; doi: https://doi.org/10.5935/abc.20170139\u003c/li\u003e\n\u003cli\u003eCattaruzza M, Słodowski W, Stojakovic M, Krzesz R, Hecker M. Interleukin-10 induction of nitric-oxide synthase expression attenuates CD40-mediated interleukin-12 synthesis in human endothelial cells. J Biol Chem. 2003; doi: https://doi.org/10.1074/jbc.M301670200\u003c/li\u003e\n\u003cli\u003eFreels JL, Nelson DK, Hoyt JC, Habib M, Numanami H, Lantz RC, Robbins RA. Enhanced activity of human IL-10 after nitration in reducing human IL-1 production by stimulated peripheral blood mononuclear cells\u003cem\u003e.\u003c/em\u003e J Immunol. 2002; doi: https://doi.org/10.4049/jimmunol.169.8.4568\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Methylene Blue, Septic Shock, Lactate, Nitric Oxide, Cytokines","lastPublishedDoi":"10.21203/rs.3.rs-3971422/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3971422/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eMethylene blue (MB) has been used to increase blood pressure in patients with septic shock by acting on guanylate cyclase and nitric oxide synthase (NOS).\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo determine whether the administration of MB to patients in the initial phase of septic shock leads to a reduction in the use of vasopressors compared to that in the control group.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis was a 1:1 randomized clinical trial of two groups (methylene blue and control). We used MB after fluid replacement, vasopressors and antibiotic therapy. Patients received a loading dose of MB (3 mg/kg) and maintenance (0.5 mg/kg/h) for 48 hours. Vasopressor doses, laboratory test results, inflammatory and anti-inflammatory cytokine levels, and hemodynamic monitoring were recorded before the infusion of MB (T1) and after 20 minutes (T2), 2 hours (T3), 24 hours (T4), 48 hours after the infusion started (T5) and 24 hours after weaning (T6).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMethylene blue therapy started within 72 hours of septic shock. The methylene blue group showed an immediate reduction in NOR dosage, earlier reduction in VAS dosage, and higher IL-10 levels compared to the control group. Integrative network analysis highlighted NO and IL-10's roles in coordinating correlations with \"Hemodynamic Monitoring\" in the control and methylene blue groups, respectively.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eEarly methylene blue (MB) administration alongside standard septic shock treatment reduces vasopressor doses, possibly involving nitric oxide (NO) mechanisms. A possible mechanism of action may involve modulation of inflammatory and anti-inflammatory mediators, enhancing immune response. However, larger and longer studies are needed for validation.\u003c/p\u003e\u003ch2\u003eTrial registration:\u003c/h2\u003e \u003cp\u003eClinical registration: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ensaiosclinicos.gov.br/rg/RBR-96584w4\u003c/span\u003e\u003cspan address=\"https://ensaiosclinicos.gov.br/rg/RBR-96584w4\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e","manuscriptTitle":"Methylene blue therapy versus standard treatment for acute-phase septic shock: a pilot randomized controlled trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-28 19:40:43","doi":"10.21203/rs.3.rs-3971422/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a3be9914-28d8-40fd-8206-ef099f0d031e","owner":[],"postedDate":"February 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-22T12:30:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-28 19:40:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3971422","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3971422","identity":"rs-3971422","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}