Methylene Blue and Blood Transfusion in Hemorrhagic Shock Resuscitation: An Experimental Porcine Study

Abstract

Introduction: Hemorrhagic shock requires immediate treatment to prevent mortality and organ dysfunction. This study evaluates the efficacy of methylene blue (MB) with blood transfusion (BT) as a potential rescue therapy in acute severe bleeding in pigs.

Methods: Thirty animals were randomly assigned to one of six groups following the induction of fixed-pressure hemorrhagic shock, after reaching a mean arterial pressure (MAP) of 55 mmHg - Group 1 (60 BT: BT after 60 minutes), Group 2 (60 MB: MB infusion after 60 minutes), Group 3 (60 MB + BT: MB and BT after 60 minutes), Group 4 (15 MB + BT: MB and BT after 15 minutes), Group 5 (15 BT + 60 MB: BT after 15 minutes and MB infusion after 60 minutes), and Group 6 (15 MB + 60 BT: MB infusion after 15 minutes and BT after 60 minutes). Hemodynamic and blood gas parameters were meticulously recorded, reversal of the shock was considered when MAP reached 90% of the baseline MAP.

Results: Except for Group 2, all groups reverted from the shock. However, groups that received MB in combination with BT, specifically Groups 3 and 4, exhibited statistically significant higher ratios of maximum MAP to baseline MAP.

Conclusion: Using MB concomitant with BT allowed the reversal of hemorrhagic shock with higher median arterial pressure levels compared to BT alone or applying MB separately from BT. This suggests that simultaneous application of MB and BT could be a more effective strategy for reversing the effects of severe acute bleeding.

Keywords: Animal Model; Bleeding Control; Blood Transfusion.; Circulatory Shock; Hemorrhagic Shock; Methylene Blue.

Fig. 1

Baseline mean arterial pressure (MAP) of each animal in the different experimental groups. There was no statistical difference between the baseline MAP of the experimental groups. Blue (Group 1 60 BT), red (Group 2 60 MB), green (Group 3 60 MB + BT), black (Group 4 15 MB + BT), brown (Group 5 15 BT + 60 MB), and violet (Group 6 15 MB + 60 BT). Kruskal-Wallis nonparametric statistic = 4.573, with P > 0.45. BT=blood transfusion; MB=methylene blue.

Fig. 2

Time course of MAP values during the experiment. Arrows mark the intervention time for groups that received blood transfusion (BT) or methylene blue (MB). The dotted line represents de mean baseline values of MAP before the shock. Blue (Group 1 60 BT), red (Group 2 60 MB), green (Group 3 60 MB + BT), black (Group 4 15 MB + BT), brown (Group 5 15 BT + 60 MB), and violet (Group 6 15 MB + 60 BT).

Fig. 3

The ratio of maximum achieved pressure to baseline pressure between the experimental groups. Reversal of shock was considered when, after shock induction, the animal’s mean arterial pressure (MAP) reached > 90% of its baseline. The concomitant infusion of MB and blood led to a tendency for higher blood pressure levels compared to blood transfusion alone or the separate use of MB. MB alone did not lead to shock reversal. Group 1 (60 BT), Group 2 (60 MB), Group 3 (60 MB + BT), Group 4 (15 MB + BT), Group 5 (15 BT + 60 MB), and Group 6 (15 MB + 60 BT). Kruskal-Wallis nonparametric statistic = 15.914, with P = 0.007094 < 0.01. BT=blood transfusion; MB=methylene blue.

Fig. 4

A) Time course of the median of PAP values during the experiment. Arrows mark the intervention time for groups that received blood transfusion (BT) or methylene blue (MB). The dotted line represents the mean baseline values of PAP before the shock. B) Relationship between the maximal PAP during the experiment and its baseline values among animals of each experimental group. There is no statistical difference between the groups. Blue (Group 1 - 60 BT), red (Group 2 - 60 MB), green (Group 3 - 60 MB + BT), black (Group 4 - 15 MB + BT), brown (Group 5 - 15 BT + 60 MB), and violet (Group 6 - 15 MB + 60 BT).

Supp. Fig. 1

A) Time course of the median of pulmonary capillary pressure (PCP) values during the experiment. Arrows mark the intervention time in groups that received blood transfusion (BT) or methylene blue (MB). The dotted line represents de mean baseline values of PCP before the shock. B) Relationship between the maximal PCP during the experiment and its baseline value among animals of each experimental group. Group 2 presents lower PCP values compared to the other groups. Kruskal-Wallis nonparametric statistic = 13.57, with P = 0.0186 < 0.05. Blue (Group 1 - 60 BT), red (Group 2 - 60 MB), green (Group 3 - 60 MB + BT), black (Group 4 - 15 MB + BT), brown (Group 5 - 15 BT + 60 MB), and violet (Group 6 - 15 MB + 60 BT).

Supp. Fig. 2

A) Time course of the median of central venous pressure (CVP) values during the experiment. Arrows mark the intervention time in groups that received blood transfusion (BT) or methylene blue (MB). The dotted line represents de mean baseline values of CVP before the shock. B) Relationship between the maximal CVP during the experiment and its baseline value among animals of each experimental group. Group 2 presents lower CVP values than the other groups. Kruskal-Wallis nonparametric statistic = 15.22, with P = 0.0094 < 0.01. Blue (Group 1 - 60 BT), red (Group 2 - 60 MB), green (Group 3 - 60 MB + BT), black (Group 4 - 15 MB + BT), brown (Group 5 - 15 BT + 60 MB), and violet (Group 6 - 15 MB + 60 BT).

Supp. Fig. 3

A) Time course of the median of cardiac output (CO) values during the experiment. Arrows mark the intervention time in groups that received blood transfusion (BT) or methylene blue (MB). The dotted line represents the mean baseline values of CO before the shock. B) Relationship between the maximal CO during the experiment and its baseline value among animals of each experimental group. There is no statistical difference between the groups. C) Time course of the median of cardiac index (CI) values during the experiment. Arrows mark the intervention time in groups that received BT or MB. The dotted line represents the mean baseline values of CI before the shock. D) Relationship between the maximal CI during the experiment and its baseline value among animals of each experimental group. There is no statistical difference between the groups. Blue (Group 1 - 60 BT), red (Group 2 - 60 MB), green (Group 3 - 60 MB + BT), black (Group 4 - 15 MB + BT), brown (Group 5 - 15 BT + 60 MB), and violet (Group 6 - 15 MB + 60 BT)

Supp. Fig. 4

A) Time course of the median of systemic vascular resistance (SVR) values during the experiment. Arrows mark the intervention time in groups that received blood transfusion (BT) or methylene blue (MB). The dotted line represents the mean baseline values of SVR before the shock. B) Relationship between the maximal SVR during the experiment and its baseline value among animals of each experimental group. There is no statistical difference between the groups. C) Time course of the median of systemic vascular resistance index (SVRI) values during the experiment. Arrows mark the intervention time in groups that received BT or MB. The dotted line represents the mean baseline values of SVRI before the shock. D) Relationship between the maximal SVRI during the experiment and its baseline value among animals of each experimental group. There is no statistical difference between the groups. Blue (Group 1 - 60 BT), red (Group 2 - 60 MB), green (Group 3 - 60 MB + BT), black (Group 4 - 15 MB + BT), brown (Group 5 - 15 BT + 60 MB), and violet (Group 6 - 15 MB + 60 BT).

Supp. Fig. 5

A) Time course of the median of pulmonary vascular resistance (PVR) values during the experiment. Arrows mark the intervention time in groups that received blood transfusion (BT) or methylene blue (MB). The dotted line represents the mean baseline values of PVR before the shock. B) Relationship between the maximal PVR during the experiment and its baseline value among animals of each experimental group. There is no statistical difference between the groups. C) Time course of the median of pulmonary vascular resistance index (PVRI) values during the experiment. Arrows mark the intervention time in groups that received BT or MB. The dotted line represents the mean baseline values of PVRI before the shock. D) Relationship between the maximal PVRI during the experiment and its baseline value among animals of each experimental group. There is no statistical difference between the groups. Blue (Group 1 - 60 BT), red (Group 2 - 60 MB), green (Group 3 - 60 MB + BT), black (Group 4 - 15 MB + BT), brown (Group 5 - 15 BT + 60 MB), and violet (Group 6 - 15 MB + 60 BT).

Supp. Fig. 6

A). Initial and final plasma nitrite and nitrate (NOx) dosages values during the experiment. B) Relationship between the final dosage of NOx and its initial dosage among animals of each experimental group. There is no statistical difference between the groups. Blue (Group 1 - 60 BT), red (Group 2 - 60 MB), green (Group 3 - 60 MB + BT), black (Group 4 - 15 MB + BT), brown (Group 5 - 15 BT + 60 MB), and violet (Group 6 - 15 MB + 60 BT). BT=blood transfusion; MB=methylene blue.

Supp. Fig. 7

A) Initial and final malondialdehyde (MDA) dosages during the experiment. B) Relationship between the final dosage of MDA and its initial dosage among animals of each experimental group. There is no statistical difference between the groups. Blue (Group 1 - 60 BT), red (Group 2 - 60 MB), green (Group 3 - 60 MB + BT), black (Group 4 - 15 MB + BT), brown (Group 5 - 15 BT + 60 MB), and violet (Group 6 - 15 MB + 60 BT). BT=blood transfusion; MB=methylene blue.