Sepsis and its implications on vasoregulation-

Sepsis is a condition with life-threatening organ dysfunction due to a dysregulated host response to infection. Among other systemic abnormalities, it can be explained by endothelial dysfunction that causes increased vascular permeability, aberrant nitric oxide (NO) metabolism, and vasodilation.

With such drastic systemic implications, it is shown to have a mortality rate between one in three and one in six of those it affects. More favorable outcomes can be seen from interventions in the initial hours following sepsis onset.

 The Surviving Sepsis Campaign (SSC) Guidelines recommend early utilization of vasopressors to maintain organ perfusion after the completion of adequate fluid resuscitation⁽¹⁾. The most used vasopressor is noradrenaline, but excess doses increase the threat of side effects like tachyarrhythmia, cardiac dysfunction, peripheral ischemia, and even immunosuppression. Large datasets have shown that a greater catecholamine vasopressor dose is linked to a higher risk of multiple organ failure and septic shock-related mortality; thus, the cumulative dose of vasopressor requirement is a justified intermediate patient-centered outcome to pave the way for adding catecholamine-sparing agents to a multimodal strategy⁽²⁾.

A combination of drugs focusing on multiple mechanisms involved in controlling blood pressure and endothelial function has been popularized in the recent past. In addition to restoring tissue perfusion, this "multimodal strategy" could additionally reduce the potential toxicity of individual drugs⁽³⁾

 Refractory shock -

Atrial natriuretic peptide and NO are endogenous vasodilators that cause myosin to be dephosphorylated at the vascular level. Competitively binding to the enzyme-soluble guanylate cyclase (sGC), NO becomes active. When this heterodimeric enzyme is activated, cyclic guanosine monophosphate (cGMP) is produced. This cGMP then activates protein kinase G, promoting the absorption of calcium, the dephosphorylation of myosin, and the relaxation of smooth muscle.

 There is a dysregulated and continuous release of nitric oxide during sepsis, caused by the vascular endothelium’s inducible nitric oxide synthase (iNOS). The relaxation of smooth muscle causes severe vasoplegia , characteristic of septic shock.

Nitric oxide also hinders the myocardial cells’ ability to utilize ATP, which lowers inotropy and reduces cardiac output. These pathological derangements together give rise to low blood pressure and, in some cases, can result in unresponsive hypotension that does not react to adrenergic medications⁽⁴⁾

Refractory shock is characterized by the requirement of vasopressor doses surpassing 0.2–0.5 mcg/kg/min of norepinephrine equivalents, or even higher. Additionally, there is a cardiac index of at least 2.2 L/ min/m² and difficulties in sustaining mean arterial pressure (MAP) above 65 mmHg.

For sepsis-induced refractory shock, non-adrenergic medications such as corticosteroids, vasopressin, and angiotensin II are being utilized. These medications have a catecholamine-sparing action, allowing for a reduction in the amount of catecholamines needed to maintain the targeted blood pressure.⁽⁵⁾

 However, there is no strong data to conclusively show that these non-adrenergic drugs are beneficial at improving morbidity and mortality outcomes.

The role of methylene blue -

There are two types of nitric oxide synthase(NOS) : inducible (iNOS), which is found in smooth muscle, endothelium, and immunomodulatory cells, and constitutive (cNOS), which is continuously active and found in neuronal and endothelial cells. 

Endotoxins and cytokines, including interleukin (IL)-1, IL-6, tumor necrosis factor-α, and interferon γ, cause the synthesis of iNOS. Sepsis patients produce these mediators at higher concentrations.

While iNOS is not subject to a negative feedback mechanism, the activity of cNOS is. Vasodilation results from increased NO synthesis in severe inflammatory conditions due to increased iNOS expression in the vascular smooth muscle and endothelium. The fact that iNOS can produce NO 1000 times more easily than cNOS accounts for its enhanced effects.

In addition, iNOS irreversibly binds to calmodulin, blocking its interaction with Ca2+ and preventing smooth muscle contraction. Further mechanisms responsible for the loss of vascular tone include the activation of ATP-sensitive potassium channels (KATP channels) in the plasma membrane of vascular smooth muscle and deficiency of the hormone vasopressin.

NO synthase is directly inhibited by methylene blue. By attaching to the iron heme moiety of sGC and halting the buildup of cGMP, it also inhibits the enzyme sGC.Vascular tone is restored and vessel reactivity to cGMP-dependent vasodilators is decreased by methylene blue by competitively inhibiting the target enzyme of NO. 

NO is not the only substrate that can activate sGC. Interleukins and oxygen-free radicals can also do so, causing vascular hyporeactivity even in the absence of NO.

Thus, the unique ability of methylene blue to inhibit sGC, the rate-limiting enzyme in the enzymatic cascade, may explain its superior ability to restore vascular tone even in the absence of NO⁽⁶⁾.

Role of methylene blue on the inflammatory process

One of the major mechanisms underlying the beneficial effects of methylene blue is its capacity to decrease the production of reactive oxygen species (ROS) from the mitochondrial electron transport chain. Therefore, methylene blue is also used as one of the mitochondrial targeted antioxidants. Increasing evidence has suggested that methylene blue can also produce beneficial effects by its anti-inflammation capacity. Methylene blue can also attenuate ischemia-reperfusion injury see in a rat model of lung transplantation by decreasing several indices of inflammation

Therefore, it is significant to discover new agents that can effectively modulate the levels of cytokines and other key components in the inflammatory processes. IL-6 plays crucial

roles in multiple pathological processes including cytokine storm that mediated many pathological processes like sepsis ⁽⁷⁾

The burden of Abdominal Sepsis

The abdomen has been associated to unacceptably high rates of morbidity and death and is the second most common site of sepsis. 

“Abdominal sepsis” is now defined as an increase of the SOFA score of ≥ 2 points due to intra-abdominal infection. Identification as well as management remain almost similar except for utilization of surgical interventions for source control.

Risk factors like bowel handling, delayed feeding and consequent impairment of gut function predispose this patient population to increased capillary leakage, which is one of the major pathological derangements of sepsis

More recent randomized studies that strictly implemented protocol-based resuscitation therapy found between 18% and 30% hospital mortality for severe sepsis, in contrast to "historical" data that claimed 40–60% hospital mortality. However, the rate of both morbidity and death is still too high

Vasoactive Inotropic score

The VIS score quantifies the amount of cardiovascular support and includes dopamine, dobutamine, epinephrine, milrinone, vasopressin and noradrenaline.This score has been utilized In the pediatric populations to accurately describe the cardiac dysfunction in both major cardiac surgeries as well as sepsis scenarios.It has recently shown to predict outcomes in adult patients with sepsis presenting to the emergency department in a fashion similar to the SOFA score ⁽⁸⁾

Our hypotheisis is that in physiological conditions, L-arginine undergoes conversion into nitric oxide through the action of a constitutive isoform of nitric oxide synthase (cNOS). In cases of sepsis, certain inflammatory mediators, such as endotoxins and cytokines like tumor necrosis factor alpha and interleukin 2, can trigger the expression of  induced nitric oxide synthase (iNOS). This results in a sustained production of nitric oxide that is unresponsive to the body’s natural negative feedback mechanisms. Consequently, nitric oxide contributes significantly to vasodilation and hypotension in the context of septic shock. Elevated nitric oxide levels can be linked to reduced responsiveness to vasopressor therapy, changes in the distribution of regional blood flow, increased capillary leakage, and the onset of multiple organ dysfunction.

Some research studies and case reports have already demonstrated the benefits of using methylene blue as an adjunct in treating vasodilation caused by septic shock. This is due to its inhibitory effect on nitric oxide-mediated vasodilation. However, it’s worth noting that most of these studies were conducted during the severe sepsis stage when patients were already receiving multiple vasopressors. To mitigate the adverse effects of vasopressors, there is a need for drugs that possess vasopressor-sparing effects.

 Hence, we aim to investigate the impact of methylene blue in the early phase of septic shock in intraabdominal sepsis patients with the goal of reducing their overall requirement for vasopressors.

We designed this RCT to assess if early administration of methylene blue could reduce the mean VIS (vasoactive-inotropic score) (calculated over 7 days from time of administration), as compared to placebo.

Primary objective – To compare the change in mean VIS (vasoactive inotropic score) in both groups over the first seven days after enrollment into the study or until discharge from the ICU, whichever occurs earlier

Randomization & blinding- All recruited patients will be randomized based on computer generated random number sequence (www.randomizer.org) into one of two groups:

Intervention group- will follow the methylene blue administration protocol.

Control group- will receive placebo.

Allocation concealment will be done by sealed envelope method. The ICU team will receive and open the envelope. The methylene blue administration protocol will be followed for the intervention group and administration of placebo for the control group respectively.

The data and outcomes will be noted by an investigator, that is not a part of the ICU team.

Recruitment of patients – All the eligible patients must fulfill the criteria of septic shock.

Refractory shock will be defined as a state of vasoplegia that requires the addition of a second vasopressor as the noradrenaline infusion rate crosses ≥ 15 mcg/min to maintain a mean arterial pressure (MAP) of 60-70 mmHg (70-80 mmHg in known hypertensives), even after adequate fluid resuscitation.

We will be studying the effects of early administration of methylene blue and thereby will administer the drug when the noradrenaline infusion rate crosses >10mcg/min

A member of the research team will talk to the patient’s relative and explain the study and intervention, its benefits as well as side effects. They will be given sufficient time to understand and sign the consent form.Recruitment will be complete only after obtaining consent.

Intervention group-

Drug- Methylene blue

Dosage- These patients will receive an intravenous (IV) infusion of 100 mg of MB in 100 ml of 0.9% sodium chloride solution over 6 h

Frequency- once daily for a total of 3 doses.

Timing- This will be given when noradrenaline infusion rate crosses ≥ 10 mcg/min to maintain a mean arterial pressure (MAP) of 60-70 mmHg (70-80 mmHg in known hypertensives), even after adequate fluid resuscitation

Collection of blood sample for IL 6 and CRP determination – Three samples will be taken:

1)  Baseline sample (before administration of methylene blue)

2)  Second sample (after second dose of methylene blue)

3)  Third sample (after third dose of methylene blue)

 Control group –These patients will receive the same volume of 100 ml of 0.9% sodium chloride without Methylene blue (placebo).

Similar sampling protocol for IL 6 and CRP will be followed.

 Critical care physicians will be responsible for the administration of intervention/placebo. Patients, clinicians, investigators and outcome assessors will be blinded to the treatment received.

Utilization of dynamic tests for fluid resuscitation:

In our ICU, fluid resuscitation of septic shock is guided by dynamic tests for prediction of volume responsiveness before any IV fluid load; the methods include the following

Passive leg raising test: The test involves elevating the legs of the patient with the head up position at 45 degrees which in turn causes increases venous return to the heart, thereby increasing preload. In a patient who is fluid-responsive, this increased preload results in an increase in cardiac output. If the patient is already adequately filled with fluids, the response will be minimal. The change in cardiac output both prior and after the positional maneuver is measured via aortic VTI (cut-off 10%)

Pulse pressure variation: It assesses the variation in pulse pressure during a complete respiratory cycle. It is calculated as the difference between the maximum and minimum pulse pressure values during a single breath divided by the average pulse pressure. In a patient who is responsive to changes in preload (fluid bolus), there will be greater variation in pulse pressure with changes in intrathoracic pressure during the respiratory cycle (cut-off 13%)

Systolic pressure variation : SPV is a measure of the variation in systolic blood pressure that occurs during a complete respiratory cycle. In fluid-responsive patients, there will be greater variation in systolic blood pressure with changes in intrathoracic pressure during mechanical ventilation. An increase in systolic blood pressure during inspiration and a decrease during expiration suggest that the patient may benefit from additional fluid administration (cut off 10%).

Tidal volume challenge test:  This test improves the reliability of PPV during low tidal volume ventilation. It involves transiently increasing tidal volume from 6 ml/kg PBW to 8 ml/kg PBW for one minute and observing the change in PPV from baseline to that at 8 ml/kg PBW ( cut off value of 2.5%)

IVC diameter and respiratory variation: The respiratory variation in IVC diameter is used as a non-invasive indicator of fluid responsiveness. In patients with normal cardiac function and compliance, an increase in IVC diameter during inspiration(on mechanical ventilation ) indicates that the right atrium is filling with blood, suggesting fluid responsiveness. If there is minimal or no change in IVC diameter, the patient will be less likely to benefit from additional fluid. The converse is true for spontaneous ventilation(IVC distensibility >18%)

Lung ultrasound : It can help assess the presence and extent of pulmonary edema seen in the form of B lines , which is the accumulation of fluid in the lung tissue. This information can act as an end point for fluid resuscitation.

We will define adequate fluid resuscitation as at least 500 ml bolus of balanced crystalloid followed by negative volume responsiveness by at least 2 different methods.

Administration of second vasopressor and corticosteroids:

In patients of both groups, adjunctive vasopressin will be initiated at a dose of 0.03 IU/min if noradrenaline dose reaches ≥ 0.2 mcg/kg/min; evaluation of volume responsiveness will be repeated as seen clinically fit if vasopressors are needed. Hydrocortisone at 200 mg/day  is also a standard in our ICU and will be withheld within 6-h after discontinuation of all vasopressors without tapering.

Tapering of vasopressor support:

Vasopressor tapering will consist of noradrenaline titration at 15–20-min intervals to maintain mean arterial pressure (MAP) between 60 and 70 mmHg (between 70 and 80 mmHg MAP for known hypertensives) until complete discontinuation while vasopressin will be progressively withdrawn by 0.005 UI/min each hour only after complete discontinuation of norepinephrine.

 Data Collection.

The following data will be collected for the patients

Baseline data- The demographic, clinical, laboratory and ventilatory information will be collected for each patient.

VIS and SOFA scores will be computed daily over 7 days or till ICU discharge, whichever occurs earlier

Outcome data- Mean VIS score over 7 days of enrollment into the study, time to vasopressor discontinuation, IL 6 and CRP levels, number of vasopressor-free days at 30 days, all-cause mortality at 30 days, days on mechanical ventilator, length of stay in ICU and hospital and change in SOFA score after intervention over 7 days.

Sample size calculation

Study by Iskender KARA et al reported that in septic shock patients, mean VIS score≥10 was seen in 49% patients. At 95% confidence level and 80% power, expecting minimum difference of 25% as significant after giving methylene blue in one group and controls group, sample size was calculated as 57 per group using the formula

Statistical analysis

The collected data will be entered in Microsoft Excel and then will be analysed and statistically evaluated using SPSS-25 version.

Normality of each variable will be assessed by using the Kolmogorov- Simirnov test. Quantitative data will be expressed by mean, standard deviation or median with interquartile range and depends on normal distribution, difference between two groups will be tested by student t test or Mann Whitney U test while for pre-post comparison paired t test or Wilcoxon signed ran test will be used. Qualitative data will be expressed in percentage and difference between the proportions will be compared by chi square test or Fisher’s exact test. ‘P’ value less than 0.05 would be considered statistically significant

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