Cardio-cerebral and metabolic effects of methylene blue in hypertonic sodium lactate during experimental cardiopulmonary resuscitation

Resuscitation (2007) 75, 88—97

EXPERIMENTAL PAPER

Cardio-cerebral and metabolic effects of methylene blue in hypertonic sodium lactate during experimental cardiopulmonary resuscitation夽 Adriana Miclescu a,∗, Samar Basu b, Lars Wiklund a a

Departments of Surgical Sciences/Anaesthesiology and Intensive Care, Faculty of Medicine, Uppsala University, S-751 85 Uppsala, Sweden b Public Health and Caring Sciences/Clinical Nutrition and Metabolism, Faculty of Medicine, Uppsala University, S-751 85 Uppsala, Sweden Received 20 February 2007 ; received in revised form 19 March 2007; accepted 23 March 2007 KEYWORDS Cardiopulmonary resuscitation; Dextran; Hypertonic solutions; Sodium lactate; Methylene blue; Oxidative injury

Summary Background: Methylene blue (MB) administered with a hypertonic-hyperoncotic solution reduces the myocardial and cerebral damage due to ischaemia and reperfusion injury after experimental cardiac arrest and also increases short-term survival. As MB precipitates in hypertonic sodium chloride, an alternative mixture of methylene blue in hypertonic sodium lactate (MBL) was developed and investigated during and after cardiopulmonary resuscitation (CPR). Methods: Using an experimental pig model of cardiac arrest (12 min cardiac arrest and 8 min CPR) the cardio-cerebral and metabolic effects of MBL (n = 10), MB in normal saline (MBS; n = 10) or in hypertonic saline dextran (MBHSD; n = 10) were compared. Haemodynamic variables and cerebral cortical blood flow (CCBF) were recorded. Biochemical markers of cerebral oxidative injury (8-iso-PGF2 ␣), inflammation (15-keto-dihydro-PGF2 ␣), and neuronal damage (protein S-100␤) were measured in blood from the sagittal sinus, whereas markers of myocardial injury, electrolytes, and lactate were measured in arterial plasma. Results: There were no differences between groups in survival, or in biochemical markers of cerebral injury. In contrast, the MBS group exhibited not only increased CKMB (P < 0.001) and troponin I in comparison with MBHSD (P = 0.019) and MBL (P = 0.037), but also greater pulmonary capillary wedge pressure 120 min after return of spontaneous circulation (ROSC). Lactate administration had an alkalinizing effect started 120 min after ROSC.

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A Spanish translated version of the summary of this article appears as Appendix in the final online version at 10.1016/j.resuscitation.2007.03.014. ∗ Corresponding author. Tel.: +46 18 6114683. E-mail address: [email protected] (A. Miclescu). 0300-9572/$ — see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.resuscitation.2007.03.014

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Conclusions: Methylene blue in hypertonic sodium lactate may be used against reperfusion injury during experimental cardiac arrest, having similar effects as MB with hypertonic saline-dextran, but in addition better myocardial protection than MB with normal saline. The neuroprotective effects did not differ. © 2007 Elsevier Ireland Ltd. All rights reserved.

Introduction Although controversy still exists concerning fluid management in humans during cardiac arrest and reperfusion, some evidence from animal studies supports the use of intravenous fluid administration1—4 during cardiopulmonary resuscitation (CPR). Hypertonic solutions administered during CPR enhance myocardial blood flow and increase resuscitation success and subsequent survival.2 In addition, the benefits of small volume resuscitation during experimental porcine CPR by use of a hypertonic-hyperoncotic solution coadministered with methylene blue (MB) up to 50 min after restoration of spontaneous circulation (ROSC) are associated with an increase in short-term survival5 and fewer signs of cerebral and cardiac injury. The protective effect of methylene blue after global ischaemia is considered to be due to inhibition of nitric oxide synthesis6—9 and inhibition of superoxide generation.10 Because MB precipitates in sodium chloride solutions and therefore the administration may be delayed during the acute settings of cardiac arrest, a mixture of hypertonic sodium lactate with MB (MBL) was developed and its cardio-cerebral and metabolic effects investigated during and after CPR.

Material and methods The regional Review Board for Animal Experimentation in Uppsala approved this prospective, randomised, laboratory animal study.

Animals Thirty Swedish domestic piglets aged 10—12 weeks with a weight of 24.8 ± 1.7 kg were obtained from a single provider. The piglets were fasted before the experiment but had free access to water. The 30 piglets were randomly allocated into three study groups.

Anaesthesia and fluid administration General anaesthesia was induced by intramuscular injection of 6 mg kg−1 Zoletil® (tiletaminezolazepam, Reading, France) combined with 2.2 mg

kg−1 Rompum® (xylazine, Bayer, Germany) and atropine 0.04 mg kg−1 . All the piglets received an intravenous injection of 20 mg morphine (Morfin Bioglan® , Uppsala, Sweden). Absence of motor response to painful stimuli before tracheotomy was the sign of adequate anaesthesia and if needed, 100 mg ketamine (Ketaminol® , Veterinaria AG, Switzerland) was added as an intravenous bolus to achieve surgical anaesthesia. Anaesthetic depth, analgesia and muscle relaxation was maintained during the entire procedure with an intravenous infusion of 8 mg kg−1 h−1 pentobarbital (Apoteket, Sweden), morphine 0.5 mg kg−1 h−1 and 0.25 mg kg−1 h−1 pancuronium bromide (Pavulon® , Organon, Netherlands). The animals were mechanically ventilated (Servo 900C, Siemens-Elema, Solna, Sweden) with a 30/70 mixture of oxygen/nitrous oxide during the preparation and 30% oxygen in air after the preparation. The ventilation was volume-controlled with an I:E ratio of 1:2, a fixed frequency of 25 min−1 and a minute volume set to yield a PaCO2 between 5.0 and 5.5 kPa. A PEEP of 5 cm H2 O was applied. The capnogram and peripheral oxygen saturation were displayed continuously (CO2 SMO Plus-8100, Novametrix, Wallingford, CT, USA) as were leads II and V5 of the electrocardiogram. All animals received fluid replacement with acetated Ringer solution (Ringer-acetat® , Fresenius Kabi, Stockholm, Sweden) as follows: 30 mL kg−1 during the first hour of preparation and thereafter a continuous infusion of 10 mL kg−1 h−1 .

Surgical preparation An 18-G arterial catheter was advanced into the aortic arch via a branch of the right external carotid artery for withdrawal of blood samples and measurement of blood pressure. A 14-G saline filled double lumen catheter was placed into the right atrium via a cutdown of the right external jugular vein to measure right atrial pressure and for drug administration. A central venous line and a 7 Fr Swan-Ganz catheter were inserted through the internal jugular vein and into the pulmonary artery, respectively. The bone lamella corresponding to the frontal sagittal sinus was resected over an area measuring 2 cm × 3 cm directly above the sagittal sinus. A 22-

90 Gauge cannula was inserted into the sagittal sinus for measurement of sagittal sinus pressure (SSP) and blood sampling. A 10-mm burr hole was made between the middle and coronal sutures of the right hemisphere and the dura was opened to place a fiberoptic flow probe with a diameter of 0.25 mm (MT B500-43, Periflux PF 2B Laser-Doppler flowmeter, Perimed, Stockholm, Sweden) on the surface of the right frontal cerebral cortex. The probe was then fixed by suturing dura and skin. The bladder was catheterised for urine collection.

Measurements and samples Haemodynamic variables including ECG recordings, heart rate (HR), systemic arterial blood pressure, right atrial pressure, pulmonary artery pressure as well as the sagittal sinus pressure (SSP) were continuously displayed (Solar 8000 monitor, Marquette Medical Systems, Milwaukee, WI, USA) and recorded (Workbench 3.0, Strawberry Tree Inc., Sunnyvale, CA, USA). Cardiac output (CO) was measured as the mean of three measurements, using the thermodilution technique. Pulmonary capillary wedge pressure (PCWP) was recorded at the same points as cardiac output. As all groups received the same dose of MB, we considered it possible to measure oxygen saturation in the different blood samples collected. The following variables were measured or calculated using standard formulas: cardiac index (CI, in L/min/m2 ), oxygen extraction ratio (calculated as the ratio between arterial-sagittal vein oxygen content difference and arterial oxygen content). Cerebral cortical blood flow (CCBF) was measured by the laser-Doppler technique, recorded every 5 s by a computer, and presented as a fraction of the steady state baseline flow level before induction of ventricular fibrillation. Samples of arterial, sagittal, and pulmonary blood were taken for blood gas analysis and acid—base balance (ABL 300, Radiometer, Copenhagen, Denmark). Oxygen saturation and haemoglobin were determined simultaneously on an OSM3 Hemoximeter (Radiometer, Copenhagen, Denmark). In addition, sagittal venous samples were collected at baseline and after ROSC at 5, 15, 30, 60, 120, 180, and 240 min. The plasma obtained after centrifugation was stored at −70 ◦ C until analyzed for 8-iso-PGF2␣ , 15-keto-dihydro-PGF2␣ and protein S-100␤. Plasma obtained after centrifugation of arterial blood drawn at baseline and at 120 and 240 min after ROSC was used to determine troponin I and CKMB. Arterial blood lactate concentrations, glucose and electrolytes were also determined (ABL 700, Radiometer, Copenhagen, Denmark) at baseline and 120 min after ROSC.

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Analytical methods Plasma concentrations of isoprostane 8-iso-PGF2∝ and 15-keto-dihydro-PGF2∝ were measured according to methods previously described.11,12 Analysis of 8-iso-PGF2␣ , a major F2 -isoprostane, is of relevance in studies of oxidative injury as an index of in vivo lipid peroxidation by free radical catalysis of arachidonic acid.11 The detection limit for 8-isoPGF2␣ was 23 pmol/L. Prostaglandin F2␣ (PG F2␣ ) is one of the major prostaglandins formed at the site of inflammation and is quantified by measurement of 15-keto-dihydro-PGF2␣ with a detection limit of 45 pmol/L.12 Myocardial and cerebral damage were assessed by serum concentrations of cardiac troponin I, CK-MB and astroglial protein S-100␤. Serum S-100␤ levels, an indicator of neurological injury, were measured by an immunoluminometric assay (LIA-mat, Sangtec® 100) with a detection limit of 0.02 ng/mL and a cutoff level of <0.12 ng/mL. CK-MB and troponin I were measured with monoclonal/polyclonal mass immunoassays (ADVIA Centaur, Bayer) with normal values below 0.12 ␮g/L for troponin I and below 6 ␮g/L for CK-MB. Experimental protocol (Figure 1) After preparation, the piglets were ventilated with 30% oxygen in air and allowed to stabilise for 1 h after which baseline measurements were made. Thereafter, in order to induce ventricular fibrillation, a 50-Hz, 20—60 V alternating transthoracic current was applied via two subcutaneous

Figure 1 Timeline of the experimental procedure: After 1 h stabilisation, animals were subjected to 20 min ventricular fibrillation (VF) including 12 min cardiac arrest and 8 min cardiopulmonary resuscitation (CPR). Methylene blue with normal saline (MBS); methylene blue in combination with hypertonic saline dextran (MBHSD); methylene blue hypertonic sodium lactate (MBL); restoration of spontaneous circulation (ROSC); alternating transthoracic shock (AC); defibrillatory shocks (DC); potassium chloride (KCl).

Methylene blue in sodium lactate needle electrodes placed subcutaneously on either side of the thorax. Cardiopulmonary arrest was defined as a decrease in aortic blood pressure below 25 mm Hg and the presence of ventricular fibrillation on the electrocardiogram. Mechanical ventilation was halted at this point. After 12 min of untreated cardiac arrest, closed-chest CPR was performed with an automatic device (Lucas® , Lund, Sweden), and mechanical ventilation with 100% oxygen was resumed with the same ventilatory settings as before induction of cardiac arrest. One minute after the start of CPR the animals were randomly assigned to receive an infusion of 55 mL kg−1 h−1 normotonic saline (MBS group, n = 10), or 10 mL kg−1 h−1 of the hypertonicsaline (7.5%) dextran (6%) solution RescueFlow® (Biophausia, Uppsala, Sweden) (MBHSD group, n = 10). To both groups, 7.5 mg kg−1 h−1 methylene blue (Metyltioninklorid 10 mg mL−1 equivalent to 8.56 mg mL−1 water free MB, Apoteket, Ume˚ a, Sweden) was administered continuously. The third group received 10 mL kg−1 h−1 of a solution prepared in our pharmacy from a mixture of methylene blue and hypertonic sodium lactate (sodium lactate 0.63 M = 7%, containing 630 mmol/L Na+ , and methylene blue 0.86 g/L, pH 6.7) (MBL group, n = 10). The relative proportions between rates of administration of normal saline versus hypertonic solutions were calculated based on the relative distribution of crystalloid and colloid solutions in the intra- and extravascular fluid compartments at equilibrium 30 min to 60 min after administration.13 One minute after beginning CPR all the animals received 0.4 U kg−1 of vasopressin (Arg8 -vasopressin) as a bolus administered via the right atrial catheter. After 8 min of external chest compressions, a monophasic counter shock was delivered through defibrillation electrode pads (Medtronic Physio-Control Corp., Seattle, WA, USA) at an energy level of 200 J. If restoration of spontaneous circulation (ROSC) was not accomplished, another two defibrillatory shocks (200 J, 360 J) and a bolus injection of adrenaline (epinephrine) 20 ␮g/kg were administered (according to 2000 resuscitation guidelines). DC shocks were then applied at an energy level of 360 J over a maximum period of 5 min. CPR was discontinued if ROSC was not achieved during this time. Restoration of spontaneous circulation was defined as return of coordinated electrical activity resulting in a systolic blood pressure greater than 60 mm Hg for at least 10 consecutive minutes. After ROSC the infusions were reduced as follows: normotonic saline 16.5 mL kg−1 h−1 with methylene blue 2.25 mg kg−1 h−1 , hypertonic saline dextran 3 mL kg−1 h−1 with methylene blue

91 2.25 mg kg−1 h−1 , and methylene blue in hypertonic sodium lactate 3 mL kg−1 h1 . All the infusions were continued at these rates for 50 min after ROSC, at which time they were stopped. After 5 min of spontaneous circulation, oxygen was reset to 30%. If arterial pH was less than 7.20 or the base deficit more than 10 mmol L−1 at 5 min after ROSC, acidosis was corrected with tris buffer mixture (Tribonat® , Kabi Fresenius, Stockholm, Sweden) 1 mmol/kg and by increasing the minute ventilation. After the resuscitation phase the only intervention was the administration of dobutamine (Dobutrex® ) in a solution of 12.5 mg/ml starting at 5 ␮g kg−1 min−1 when the systolic blood pressure decreased to less than 70 mm Hg. Hemodynamic variables and blood gases were observed for 4 h after ROSC. After completion of the study, all animals received an injection of 10 ml potassium chloride 20 mmol/mL and were sacrificed.

Statistical analysis Data are presented as means ± S.E. Repeated measures two-way analysis of variance was used for comparisons of the groups over time after the data were shown to be normally distributed. In order to secure normally distributed data, analyses of 8-isoPGF2␣ and 15-keto-dihydro-PGF2␣ were done after logarithmic transformation. Where ANOVA showed significant differences, a Bonferroni multiple comparison test was used. Survival analysis was carried out using the method of Kaplan and Meier (GraphPad PRISM® version 4, GraphPad Software, Inc. San Diego, CA, USA), and comparisons between groups were made using the log-rank test as well as the log-rank test for trend, as survival was considered to be a variable of the ordinal type. P-values <0.05 were regarded as statistically significant.

Results There were no differences in baseline variables between groups.

Survival Survival was not different between the groups. In MBS and MBL two pigs in each group, and one in MBHSD did not achieve ROSC. There were no later deaths.

Haemodynamics (Figure 2) Following cardiac arrest, all groups presented similar systemic haemodynamic changes with an

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Figure 2 Hemodynamic variables: (A) mean arterial pressure (MAP); (B) mean arterial pulmonary pressure (PAMP); (C) pulmonary capillary wedge pressure (PCWP); (D) cardiac index (CI), during cardiac arrest (CA) and ventricular fibrillation (VF), after restoration of spontaneous circulation (ROSC) and during spontaneous circulation (SC). Symbols: methylene blue in lactate (MBL) presented as black filled circles; methylene blue in saline (MBS) represented by black filled triangles; methylene blue in hypertonic saline dextran (MBHSD) represented by white circles. PCWP was increased at 180 min after ROSC in the MBS group (P = 0.023) and remained increased until the end of the experiment. The values are presented as means ± S.E.

overshoot of arterial and pulmonary arterial blood pressures and cardiac output in all groups (P < 0.0001) 5—10 min after ROSC. After that, a hypotensive period gradually followed in all groups, with the lowest levels at approx. 30 min. The decreased blood pressure lasted approximately 1 h after ROSC, followed by a gradual increase to baseline levels. No differences between groups were seen in the number of pigs that received dobutamine. PCWP was increased at 180 min after ROSC in the MBS group (P = 0.023) and remained increased until the end of the experiment. Tendencies toward higher pressures in the pulmonary artery and right atrium were observed at 120 min after ROSC in the same group but they failed to reach statistical significance.

Cerebral cortical blood flow, cerebral perfusion pressure, cerebral oxygen extraction ratio (Figure 3) There were no differences between groups in cerebral perfusion pressure. During the early reperfusion phase (5—10 min after ROSC), the mean CCBF increased in all groups in comparison with baseline values (P < 0.001). Thereafter, this blood flow started to decrease and gradually returned to base-

line in all the groups. Differences were observed at 5 min after ROSC with an increase in CCBF in the MBS group in comparison with the MBHSD group (P = 0.023) and the MBL group (P = 0.021). At the same time point, the increased CCBF was associated with a numerical difference in the oxygen extraction ratio in the MBS group in comparison with both of the other groups, but it failed to reach statistical significance.

Oxidative, inflammatory, and neurological markers (Table 1) No differences were recorded between groups in the levels of 8-iso-PGF2␣ , 15-keto-dihydro-PGF2␣, and protein S-100␤. The sagittal sinus plasma concentration increased 16- and 3-fold with a maximum 30—60 min after ROSC for the oxidative and inflammatory markers, respectively, while the concentration maximum (5-fold increase) occurred 5—15 min after ROSC for protein S-100␤.

Myocardial enzymes (Table 2) All three groups had increased levels of myocardial enzymes indicating myocardial injury. The increase of troponin I was greater in MBS group at 240 min

Methylene blue in sodium lactate

Figure 3 Cerebral oxygen extraction ratio (COER), cerebral perfusion pressure (CePP) and cerebral cortical blood flow (CCBF). All groups displayed an overshoot in CCBF and CePP at 5 min after ROSC in comparison with baseline (P < 0.0001). There was a difference between groups at 5 min after ROSC in cerebral cortical blood flow (CCBF) that was increased in the MBS group in comparison with the MBHSD group (P = 0.023) and the MBL group (P = 0.021). Symbols: MBL presented as black filled circles; MBS represented by black filled triangles; MBHSD represented by white circles. The values are presented as means ± S.E.

in comparison with MBHSD (P = 0.019) and MBL (P = 0.037). CK-MB was also increased in this group in comparison with both of the other groups at 120 min and 240 min (P < 0.001).

Lactate, electrolytes, glucose (Table 2) The baseline blood levels of lactate in the three study groups did not differ. The lactate concentration was increased 2 h after ROSC in all groups (P < 0.05). Differences in lactate were recorded between groups, with higher levels in the MBL group only in comparison with the MBS group (P = 0.034), with an intermediate level in the MBHSD group. The sodium load administered to the MBHSD group increased the plasma sodium

0.13 0.18c 0.14c 0.10b 0.05 0.06 0.06 0.05 ± ± ± ± ± ± ± ± 0.48 2.45 2.25 1.50 0.89 0.66 0.56 0.48 0.05 0.20c 0.21c 0.12c 0.07 0.05 0.07 0.08 ± ± ± ± ± ± ± ±

MBL MBHSD MBS

0.52 2.43 2.48 1.57 0.94 0.71 0.61 0.65 0.05 0.30c 0.33c 0.19c 0.12 0.06 0.06 0.05 ± ± ± ± ± ± ± ± 0.43 2.10 2.10 1.30 0.86 0.58 0.54 0.50 36 62c 125c 209c 77c 48 91 41 ± ± ± ± ± ± ± ± 192 503 596 726 526 475 586 378 29 92c 105c 152c 146c 76 58 30 ± ± ± ± ± ± ± ±

MBL MBHSD MBS

175 364 416 538 520 371 314 250 47 127c 121c 99c 106c 74c 72 25c ± ± ± ± ± ± ± ± 254 490 706 751 706 564 461 329 0.3 6.4c 6.7c 9.6c 9.4c 5.6c 6.8b 4.0a ± ± ± ± ± ± ± ± 6.0 91.6 90.2 110 111 77.1 40.5 14.7 1.2 4.9c 11c 12c 11c 5.3c 5.5b 3.6a ± ± ± ± ± ± ± ± 7.2 82.1 102 119 116 77.8 39.6 23.5 0.6 6.5c 7.3c 4.6c 10.0c 11.5c 11.3b 7.8a ± ± ± ± ± ± ± ±

MBL MBHSD MBS

Protein S-100 ␤ (ng/mL) 15-Keto-dihydro PGF2␣ (pmol/L) 8-Iso-PGF2␣ (pmol/L) Time (min)

6.6 63.1 81.1 93.6 99.7 64.2 32.9 18.4 0 5 15 30 60 120 180 240

Indicators of neurological injury Table 1

The indicator for peroxidation injury 8-iso-PGF2␣ , the indicator of inflammation 15-keto-dihydro-8-iso-PGF2␣ , and the indicator for neurological cellular injury determined by astroglial protein S-100␤. There are no significant group differences. The values are presented as means ± S.E. a P < 0.05 in comparison with baseline. b P < 0.01 in comparison with baseline. c P < 0.001 in comparison with baseline.

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14.34 ± 9.64 7.04 ± 4.63

concentration after ROSC compared to prearrest conditions (P < 0.001), and compared to the other treatment groups (P < 0.001). Hypertonic sodium lactate administration had no effect on serum sodium concentration, while administration of normal saline decreased serum sodium 120 min after ROSC (P < 0.001). Hypertonic saline dextran (MBHSD) increased the serum chloride concentration (P < 0.001) in comparison with baseline, whereas lactate administration (MBS) substantially decreased it (P < 0.001). There was a significant difference between these two groups (P < 0.001). Calcium decreased in the MBL group in comparison with both other groups (P < 0.05) and baseline (P < 0.05). There was no effect on serum potassium or on blood glucose in any of the groups.

0 135.82 3.95 102.73 1.30 8.35 1.58 5.72 0.45 3.22 11.0 ± 8.57 6.03 ± 2.49

Effects on acid—base status (Figure 4)

0 135.05 4.05 101.53 1.34 8.97 1.78 5.87 0.77 4.36 Time (min) Sodium Potassium Chloride Calcium Glucose Lactate Anion gap Troponin I CKMB

P < 0.05 between groups. *

The values are presented as means ± S.E. ** P < 0.01 between groups. *** P < 0.001 between groups. a P < 0.05 in comparison with baseline. b P < 0.01 in comparison with baseline. c P < 0.001 in comparison with baseline.

47.06 ± 46.2c,* 18.20 ± 8.13***

0 134.13 4.12 102.15 1.37 9.22 1.82 5.86 0.37 3.18

MBSAL

± ± ± ± ± ± ± ± ±

1.94 0.28 1.43 0.06 0.82 0.26 0.92 1.0 2.06

120 133.15 4.08 102.13 1.27 8.61 2.86 5.2 25.32 14.31

± ± ± ± ± ± ± ± ±

1.64c 0.29 1.82 0.06a 1.45 0.72a 1.05 26.3 6.35***

240

MBHSD

± ± ± ± ± ± ± ± ±

1.64 0.35 1.80 0.12 1.41 0.43 1.41 0.29 0.78

120 138.91 3.82 108.14 1.37 10.54 3.42 6.14 7.38 4.61

± ± ± ± ± ± ± ± ±

1.76c,*** 0.18b 1.70c 0.11 2.22 0.99a 1.35 7.72 1.15

240

MBL

± ± ± ± ± ± ± ± ±

1.47 0.27 2.35 0.05 1.25 0.22 1.29 0.37 0.65

120 134.43 3.93 99.75 1.19 9.44 4.01 6.78 16.34 7.86

± ± ± ± ± ± ± ± ±

1.40 0.46 3.35c,*** 0.07a,* 2.27 1.24c,** 1.39a,* 16.25 3.97

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A. Miclescu et al.

Group

Table 2 Serum electrolytes, glucose, lactate, anion gap (mmol/L) and the indicators of myocardial injury, troponin I and CKMB (␮g/mL) before cardiac arrest at baseline and after return of spontaneous circulation

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The arterial blood pH gradually decreased during cardiac arrest and CPR and this was a combined metabolic and respiratory acidosis. Acidaemia was profound (pH < 7.2) in all the groups 5 min after ROSC and needed correction with tris buffer mixture as well as increased ventilation. The total dose of buffer administered to the three groups did not differ. pH returned to normal values at 120 min after ROSC in the MBL group, with a mean value that was slightly higher in comparison with the MBS (P = 0.01) and the MBHSD groups (P = 0.03). In contrast to the arterial pH, the sagittal sinus blood pH decreased immediately after commencement of CPR and started to increase at 16 min after ROSC. In parallel with the pH effects, the base excess was higher in the MBL group from 120 min until the end of the study.

Discussion In a previous study,5 we demonstrated significant neuro- and myocardial protection against reperfusion injury from cardiac arrest when methylene blue added to a hypertonic-hyperoncotic solution was used, as compared with either HSD or normal saline. This difference was judged mostly to be an effect of MB that improved coronary perfusion pressure, cardiac index, decreased myocardial and brain injury and reduced cerebral peroxidation and inflammation.5 This follow-up study compared circulatory and tissue protective effects of MB used along with different solvents/vehicles, i.e. hypertonic lactate, normal saline and HSD, respectively. Thus, the present study demonstrates that the neuroprotective effects were independent of the

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Figure 4 Effects on acid—base status. After giving MB hypertonic sodium lactate (MBL-black filled circles), the pH returned at basal levels at 120 min after return of spontaneous circulation (ROSC) and was slightly higher until the end of the experiment in comparison with the MBS group (P < .01), represented as black triangles, and the MBHSD group (P = 0.03), represented as white circles. The values are expressed as means ± S.E.

vehicle or solvent, but the myocardial protection was significantly better when MBHSD and MBL were used as compared to MB in normal saline. A modification of the protocol was that in the present study cerebral venous blood was sampled from the sagittal sinus vein, while it was taken from the internal jugular sinus in the earlier study. When comparing the results of the analyses from the two sites, it is observed that 8-iso-PGF2␣ was more than doubled in the plasma sampled in the sagittal sinus vein and it peaked later than in the plasma samples from the internal jugular vein. The magnitude of the difference between the sample sites was similar for 15-keto-dihydro-PGF2␣ but not for the astroglial protein S-100␤, where the difference was less. Interpretation of this comparison indicates that when determined in samples from the sagittal sinus, the other two indicators were more specific for cerebral injury than the astroglial protein S-100␤ in connection with experimental porcine cardiac arrest and CPR. The blood obtained from the sagittal sinus has mainly perfused the cerebrum, while the blood in the internal jugular vein has also perfused extracranial tissues. The higher levels of the indicator of peroxidized arachidonic acid in sagittal sinus corroborates with our earlier statement that cardiac arrest and ROSC lead to a

more severe state of oxidative stress in the brain compared to the rest of the body.14 Greater cardiac filling pressures and myocardial enzymes in MBS 2 h after maximal saline administration associated with appropriate urine production indicate that the cardiac malfunction was not caused by a cumulative excessive administration of saline, but the result of a less compliant left ventricle after administration of isotonic saline. In contrast, administration of hypertonic lactate has been demonstrated to improve cardiac function15 and to be well-tolerated,16 in agreement with the present findings. Systemic hypertension that occurs regularly immediately after successful resuscitation results in increased cerebral blood flow that has been suggested to be a major contributor to blood—brain barrier damage.17 The increase in CCBF in the MBS group was associated with an inverse reaction of the cerebral oxygen extraction ratio at 5 min. It can be speculated that this was a sign of a deficient distribution of the cerebral blood flow to brain regions where blood flow is profuse, and less to other regions where blood flow is limited as was demonstrated in a previous study.18 Except the administration of rapid infusion of 2-L ice-cold saline for induction of mild hypothermia,19 there are no published prospective human stud-

96 ies on the possible benefits of fluid administration after cardiac arrest and there is no human trial to support volume expansion during CPR. It is known that during CPR there is a plasma shift from the intravascular to the extravascular compartment leading to intravascular hypovolaemia and haemoconcentration.20,21 Therefore, the logical approach has been to use hypertonic-hyperoncotic fluid therapy, i.e. so-called small volume resuscitation, which might compensate for this leak. The importance of fluid infusion rates and volumes for achieving a certain plasma volume expansion has been emphasized.22 In our study HSD and hypertonic sodium lactate were given in a total dose of 3.52 ± 0.2 mL/kg during a period of 1 h, which is a comparatively slow infusion rate.23 It has been demonstrated previously that the addition of colloid does not enhance the haemodynamic effects of a hypertonic saline.2 Thus, we used the same administration rate for hypertonic saline dextran and hypertonic sodium lactate. No differences in systemic circulatory variables were found between the two groups, despite the higher sodium levels recorded 2 h after ROSC in the group that received HSD. The latter group received a higher sodium load (approx. 300 mmol) as compared with 12 mmol in the MBS group and 57 mmol in the MBL group, which is well reflected in the serum sodium concentration 120 min after ROSC. The total dose of MB administered was similar (2.7—3.4 mg/kg). Doserelated toxicity of methylene blue is described24 for doses over 2 mg/kg, but considerably higher doses have also been used without untoward effects. Methylene blue administration in a total dose of 7 mg/kg has been used in the treatment of methemoglobinaemia,25 and a very high dose was accidentally administered without toxic effects.26 Hence, it might be possible to use even higher doses of MB as a rescue treatment in cardiac arrest. The clinical efficacy of methylene blue is well documented with intermittent27,28 and continuous administration.29 The advantage of the mixture MB with lactate as described in our study is that it is a more rapid way of administration during ongoing cardiac arrest. The administration of MB with sodium chloride solutions needs two infusions sets in order to avoid precipitation, thus, more difficult to be used during resuscitation. In the present study the blood lactate concentration increased in all groups after cardiac arrest but, as expected, higher levels were observed after administration of hypertonic sodium lactate, which contains 630 mmol/L of racemic sodium lactate. The average dose of lactate was approximately 90 mL, which equals 57 mmol and is well within the dose range regularly used in haemorrhagic shock.30

A. Miclescu et al. Administration of hypertonic sodium lactate returned pH to control levels in the MBL group, whereas it remained lower in the end of the study in the other two groups. This was in accordance with previous authors who found an improvement in acidosis31 after administration of large amounts of Ringer’s lactate in haemorrhagic shock. It was demonstrated that clearance of exogenous lactate was not altered significantly in early postoperative shock accompanied by lactic acidosis.15 It should be noted that piglets normally have an extracellular pH slightly above than in humans. Exogenous administration of sodium lactate acts as a buffer because the anion lactate is also metabolised to bicarbonate, leading to a loss of free protons when CO2 is released from carbonic acid, resulting in an alkalinizing effect.15,32 As far as haemodynamics and oxygenation variables are concerned, there were no differences between the MBS group and the other groups. There are several limitations of the present study. One of the limitations is the age of animals that reflects a paediatric response to cardiac arrest, but the haemodynamic and metabolic variables used are appropriate and similar to those used during critical care settings. Second, we did not retain histological samples of the heart and brain that would be the subject of future articles.

Conclusions There were no differences in indicators of cerebral and myocardial damage or haemodynamic data for methylene blue when administered in a sodium lactate or hypertonic saline dextran solution in contrast to methylene blue in normal saline. If methylene blue can be shown to improve long-term outcome after cardiac arrest, a mixture of MB with sodium lactate offers the advantage of being stable without the precipitation that occurs in sodium chloride solutions, thus being able to be used more rapidly in the acute setting of cardiac arrest.

Conflict of interest All the authors hereby state that there is no conflict of interest.

Acknowledgements The authors are grateful to Anders Nordgren, Monika Hall, and Elisabeth Pettersson for excel-

Methylene blue in sodium lactate lent technical assistance. This work was financed by grants from the Laerdal Foundation for Acute Medicine, Stavanger, Norway.

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