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Written by Clark Strunk
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This small RCT demonstrated that a low-dose continuous infusion of neostigmine in patients with septic shock resulted in downregulation of inflammatory cytokines and suggested possible improvements in organ dysfunction, illness severity, and mortality.
A neo way to neutralize the cytokine storm in sepsis…
This was a single-center, double-blinded, randomized trial in patients with septic shock, comparing a low-dose continuous infusion of neostigmine over 5 days to placebo. Neostigmine is thought to enhance signaling via the cholinergic anti-inflammatory pathway (ChAP); consequently, the author’s primary outcome was a reduction in TNF-α levels, which occurred more in the intervention group. The most provocative finding was a significant reduction in 28-day mortality: 27% in the intervention group versus 54% in the placebo group (p = 0.02). There were also statistically significant reductions in the SOFA score and APACHE II score favoring the intervention group. Notably, there were no differences between groups in safety outcomes including bradycardia, hypotension, and heart rate trends.
How will this change my practice?
The results of this trial will not change my practice due to the small number of patients that were randomized and the lack of a patient-centered primary outcome. However, given the improvements seen in organ dysfunction, 28-day mortality NNT = 4, relatively cheap and familiar therapy, strong physiologic rationale, and no signal for safety concerns, I am hopeful for larger, multicenter trials that may reinforce these exciting results.
Source
Effect of Neostigmine on Attenuation of Proinflammatory Cytokines When Given as an Adjuvant Therapy in Septic Shock: A Randomized Control Trial. Crit Care Med. 2026 Feb 12. doi: 10.1097/CCM.0000000000007051. Epub ahead of print. PMID: 41677407.
View JournalFeed Critical Appraisal
Critical Appraisal
Study Identification
Title: Effect of Neostigmine on Attenuation of Proinflammatory Cytokines When Given as an Adjuvant Therapy in Septic Shock: A Randomized Control Trial
Authors: Mirdhu Bashni T, Nikhil Kothari, Ankur Sharma, Shilpa Goyal, Shrimanjunath Sankanagoudar, Bharat Paliwal, Pradeep Kumar Bhatia
Journal: Critical care medicine
Year: 2026
DOI: 10.1097/CCM.0000000000007051
PMID:
41677407 →
Trial Registration: CTRI/2023/07/055054
Country / Region: India
Setting: One adult ICU at a tertiary academic medical institution
Background
Sepsis is a major global health challenge with high mortality, driven by a dysregulated host response ("cytokine storm") leading to organ dysfunction. Existing immunomodulatory strategies have limited high-quality evidence. The cholinergic anti-inflammatory pathway (ChAP) offers a novel neuro-immunological approach to inflammation control, involving vagus nerve stimulation to suppress proinflammatory cytokine release. Neostigmine, an acetylcholinesterase inhibitor, enhances ChAP activation and has shown promise in experimental sepsis models. Previous human studies suggested therapeutic promise but lacked data on specific inflammatory biomarkers.
Study Question
To evaluate whether neostigmine administration modulates the inflammatory response in sepsis by enhancing cholinergic anti-inflammatory activity and to determine whether adding neostigmine to standard therapy could modulate the inflammatory response and enhance clinical outcomes in septic shock.
Study Design & Conduct
Study Design: Randomized Controlled Trial
Prospective / Retrospective: Prospective
Multicenter: No
Unit of Allocation: Patient
Unit of Analysis: Patient
Randomization Method: Simple randomization using computer-generated random numbers, implemented via sequentially numbered, opaque, sealed envelopes.
Allocation Concealment: Sequentially numbered, opaque, sealed envelopes containing coded syringes.
Blinding: Patients and outcome assessors were blinded.
Follow-up Duration: 28 days for mortality; 5 days for primary and most secondary outcomes; weekly monitoring for 1 month.
Prospective / Retrospective: Prospective
Multicenter: No
Unit of Allocation: Patient
Unit of Analysis: Patient
Randomization Method: Simple randomization using computer-generated random numbers, implemented via sequentially numbered, opaque, sealed envelopes.
Allocation Concealment: Sequentially numbered, opaque, sealed envelopes containing coded syringes.
Blinding: Patients and outcome assessors were blinded.
Follow-up Duration: 28 days for mortality; 5 days for primary and most secondary outcomes; weekly monitoring for 1 month.
Population
Inclusion Criteria:
- Adults 18–80 years old
- Diagnosis of septic shock
- Enrolled within 6 hours of diagnosis
- Need for vasopressor support to maintain MAP ≥ 65 mmHg despite adequate fluid resuscitation (30mL/kg balanced crystalloids within the first hour)
- Point-of-care ultrasound to confirm fluid responsiveness and exclude cardiogenic shock.
Exclusion Criteria:
- Known contraindications to neostigmine (e.g., bradycardia, asthma, intestinal obstruction, etc.)
- Pregnancy
- Preexisting conduction abnormalities
- Significant chronic organ dysfunction not related to sepsis.
Number Screened: 2249
Number Enrolled: 82
Number Analyzed: 82
Number Enrolled: 82
Number Analyzed: 82
Key Baseline Characteristics
Age: Neostigmine 53.6±15.3 years, Control 51.3±18.0 years (mean ± SD)
Sex: Neostigmine 26 (63%) male, Control 28 (68%) male
Disease Severity: APACHE II scores (Neostigmine 23.1±5.2, Control 21.9±5.9)
Care Setting Distribution: Adult ICU at a tertiary academic medical institution.
Sex: Neostigmine 26 (63%) male, Control 28 (68%) male
Disease Severity: APACHE II scores (Neostigmine 23.1±5.2, Control 21.9±5.9)
Care Setting Distribution: Adult ICU at a tertiary academic medical institution.
Additional Baseline Characteristics
- Hypertension (Neostigmine 13 (32%), Control 10 (24%))
- Diabetes (Neostigmine 12 (29%), Control 15 (37%))
- Chronic kidney disease (Neostigmine 3 (7%), Control 5 (12%))
- Baseline TNF-α levels (Neostigmine median 88 pg/mL, Control median 66 pg/mL, p < 0.001) - noted as a baseline imbalance.
Exposures / Interventions
Primary Exposure / Intervention
Description: Continuous intravenous neostigmine infusion.
Definition / Dose: 0.2 mg/hr (2 mL/hr of 0.1 mg/mL solution).
Timing: Administered for 5 days (120 hours), initiated within 6 hours of septic shock diagnosis.
Classification Method: Randomization
Protocolized / Discretionary: Protocolized
Description: Continuous intravenous neostigmine infusion.
Definition / Dose: 0.2 mg/hr (2 mL/hr of 0.1 mg/mL solution).
Timing: Administered for 5 days (120 hours), initiated within 6 hours of septic shock diagnosis.
Classification Method: Randomization
Protocolized / Discretionary: Protocolized
Comparator / Reference
Description: Placebo (normal saline).
Definition: Continuous intravenous normal saline infusion at 2 mL/hr for 120 hours.
Description: Placebo (normal saline).
Definition: Continuous intravenous normal saline infusion at 2 mL/hr for 120 hours.
Outcomes & Results
Primary Outcomes
Decrease in tumor necrosis factor-alpha (TNF-α) levels
Definition: Serum TNF-α concentrations.
Time Point: Baseline, 72 hours (day 3), and 120 hours (day 5).
Measurement Method: Enzyme-linked immunosorbent assay (ELISA) Kit (Abbkine), double-antibody sandwich method.
Results: Neostigmine group: Day 1 median 88 (IQR 62.73-127.51), Day 3 median 42.3 (IQR 22.1-80.5), Day 5 median 23 (IQR 9.80-65.76). Control group: Day 1 median 66 (IQR 44.35-93.65), Day 3 median 57.1 (IQR 43.95-85), Day 5 median 59.5 (IQR 31.85-91.39). p < 0.001 for TNF-α day 5. TNF-α day 5-day 1 difference: Neostigmine -56.4, Control -6.9 (p < 0.001).
Definition: Serum TNF-α concentrations.
Time Point: Baseline, 72 hours (day 3), and 120 hours (day 5).
Measurement Method: Enzyme-linked immunosorbent assay (ELISA) Kit (Abbkine), double-antibody sandwich method.
Results: Neostigmine group: Day 1 median 88 (IQR 62.73-127.51), Day 3 median 42.3 (IQR 22.1-80.5), Day 5 median 23 (IQR 9.80-65.76). Control group: Day 1 median 66 (IQR 44.35-93.65), Day 3 median 57.1 (IQR 43.95-85), Day 5 median 59.5 (IQR 31.85-91.39). p < 0.001 for TNF-α day 5. TNF-α day 5-day 1 difference: Neostigmine -56.4, Control -6.9 (p < 0.001).
Secondary Outcomes
Procalcitonin (PCT) levels
Definition: Serum PCT levels.
Time Point: Day 1, Day 3, Day 5.
Measurement Method: Not reported
Results: Neostigmine group: Day 1 median 3.6, Day 3 median 2.69, Day 5 median 1.08. Control group: Day 1 median 4.24, Day 3 median 4.88, Day 5 median 5.05. p = 0.012 for PCT day 5.
Definition: Serum PCT levels.
Time Point: Day 1, Day 3, Day 5.
Measurement Method: Not reported
Results: Neostigmine group: Day 1 median 3.6, Day 3 median 2.69, Day 5 median 1.08. Control group: Day 1 median 4.24, Day 3 median 4.88, Day 5 median 5.05. p = 0.012 for PCT day 5.
Heart Rate (HR)
Definition: Beats/min.
Time Point: Day 1, Day 3, Day 5.
Measurement Method: Not reported
Results: No statistically significant difference between groups (e.g., Day 5: Neostigmine 94.48±18.08, Control 102.63±15.47, p=0.767).
Definition: Beats/min.
Time Point: Day 1, Day 3, Day 5.
Measurement Method: Not reported
Results: No statistically significant difference between groups (e.g., Day 5: Neostigmine 94.48±18.08, Control 102.63±15.47, p=0.767).
Noradrenaline (Noradr) requirements
Definition: Microgram/kg/min.
Time Point: Day 1, Day 3, Day 5.
Measurement Method: Not reported
Results: Day 1: Neostigmine 0.35±0.22, Control 0.24±0.14 (p=0.006). Day 5: Neostigmine 0.18±0.21, Control 0.24±0.19 (p=0.213).
Definition: Microgram/kg/min.
Time Point: Day 1, Day 3, Day 5.
Measurement Method: Not reported
Results: Day 1: Neostigmine 0.35±0.22, Control 0.24±0.14 (p=0.006). Day 5: Neostigmine 0.18±0.21, Control 0.24±0.19 (p=0.213).
Vasopressin (Vaso) requirements
Definition: Units/h.
Time Point: Day 1, Day 3, Day 5.
Measurement Method: Not reported
Results: Day 5: Neostigmine 0.18±0.6, Control 1.22±1.12 (p<0.001).
Definition: Units/h.
Time Point: Day 1, Day 3, Day 5.
Measurement Method: Not reported
Results: Day 5: Neostigmine 0.18±0.6, Control 1.22±1.12 (p<0.001).
Serum Creatinine (S.Cr) levels
Definition: mg/dL.
Time Point: Day 1, Day 3, Day 5.
Measurement Method: Not reported
Results: Day 5: Neostigmine 1.29±0.69, Control 2.25±1.26 (p<0.001).
Definition: mg/dL.
Time Point: Day 1, Day 3, Day 5.
Measurement Method: Not reported
Results: Day 5: Neostigmine 1.29±0.69, Control 2.25±1.26 (p<0.001).
Sequential Organ Failure Assessment (SOFA) scores
Definition: Organ failure score.
Time Point: Day 1 to Day 5.
Measurement Method: Not reported
Results: Significant reduction from day 1 to day 5 in neostigmine group (p < 0.001).
Definition: Organ failure score.
Time Point: Day 1 to Day 5.
Measurement Method: Not reported
Results: Significant reduction from day 1 to day 5 in neostigmine group (p < 0.001).
APACHE II scores
Definition: Illness severity score.
Time Point: Baseline to Day 5.
Measurement Method: Not reported
Results: Significant decline in neostigmine group (p < 0.001), remaining unchanged in controls.
Definition: Illness severity score.
Time Point: Baseline to Day 5.
Measurement Method: Not reported
Results: Significant decline in neostigmine group (p < 0.001), remaining unchanged in controls.
28-day mortality
Definition: All-cause mortality at 28 days.
Time Point: 28 days.
Measurement Method: Not reported
Results: Neostigmine 11 (27%), Control 22 (54%) (p=0.02).
Definition: All-cause mortality at 28 days.
Time Point: 28 days.
Measurement Method: Not reported
Results: Neostigmine 11 (27%), Control 22 (54%) (p=0.02).
Risk of Bias
Risk of Bias - RoB 2
- Bias arising from the randomization process (Some concerns): Although computer-generated randomization and allocation concealment were used, there was a statistically significant baseline imbalance in the primary outcome (TNF-α levels) between the intervention and control groups (88 vs 66 pg/mL, p < 0.001), which the authors acknowledge as a limitation that 'may have influenced observed effects and complicated interpretation.'
- Bias due to deviations from intended interventions (Low): The study was double-blinded, and the authors reported no loss to follow-up and no patients switched groups or stopped treatment, indicating adherence to the intervention protocol.
- Bias due to missing outcome data (Low): The authors explicitly state there was no loss to follow-up, and an intention-to-treat analysis approach was used for all 82 randomized patients.
- Bias in measurement of the outcome (Low): Outcome assessors were blinded to group allocation. The primary outcome (TNF-α levels) was measured using a standardized ELISA kit, and other outcomes were standard clinical parameters.
- Bias in selection of the reported result (Low): The study is registered (CTRI/2023/07/055054), and primary and secondary outcomes are clearly defined in the abstract and methods, suggesting no selective reporting.
Transparency
Funding Disclosed:
TRUE
COI Statement Present: TRUE
COI Statement Present: TRUE
This critical appraisal is generated using a structured framework applied to the full text
of the original article and is presented separately from the physician’s commentary.
