Effects of a 5HT2 receptor agonist on anaesthetized pigs susceptible to malignant hyperthermia

M. U. Gerbershagen*,1, F. Wappler1, M. Fiege1, K. Kolodzie1, R. Weißhorn1, W. Szafarczyk1, C. Kudlik2 and J. Schulte am Esch1

1 Department of Anaesthesiology, University Hospital Hamburg-Eppendorf, Martinistraße 52, D-20246 Hamburg, Germany. 2 Department of Surgery, Asklepios Westklinikum Hamburg, Suurheid 20, D-22559 Hamburg, Germany

Corresponding author. E-mail: gerbershagen@uke.uni-hamburg.de

Accepted for publication: April 10, 2003


    Abstract
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 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Background. The pathophysiology of the serotoninergic system in malignant hyperthermia (MH) is not completely understood. The serotonin-2 (5HT2A) receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) induces typical MH symptoms, including skeletal muscle rigidity, an increase in body temperature, hyperventilation and acidosis in conscious MH-susceptible (MHS) pigs. Whether these symptoms are directly generated in skeletal muscle, result from central serotonergic overstimulation or from a porcine stress syndrome remains unresolved. In this study the in vivo effects of DOI on anaesthetized (and thus stress-protected) MHS and MH-normal (MHN) pigs were investigated.

Methods and results. DOI 1 mg kg–1 was administered three times at 40-min intervals to five MHS and five MHN anaesthetized pigs. Body temperature, heart rate, muscle tone, arterial carbon dioxide pressure (PaCO2), pH and creatine kinase concentrations were measured. The clinical occurrence of MH was defined by PaCO2 above 70 mm Hg and an increase in body temperature of more than 2 °C. Intragroup differences were analysed with the Friedman test as an overall non-parametric ANOVA and, in case of significance, with the Wilcoxon test. Intergroup comparisons were performed with the Mann–Whitney U-test (statistical significance P<0.05). MHS and MHN pigs developed muscle fasciculations, significant increases in body temperature and PaCO2 and a significant decrease in pH after the administration of DOI. These changes were comparable in both groups until the third dose of DOI, when in MHS pigs heart rate and PaCO2 rose significantly and pH fell significantly compared with MHN pigs. All MHS pigs fulfilled the MH criteria. Body temperature increased by more than 2 °C in all MHN pigs and PaCO2 exceeded 70 mm Hg in two. Thus, two MHN pigs fulfilled the criteria of MH.

Conclusions. The comparability of the clinical presentation following DOI administration in MHS and MHN animals and the order of the development of MH-like symptoms favour the hypothesis of a central serotonergic overstimulation, leading to a serotonin syndrome.

Br J Anaesth 2003; 91: 281–4

Keywords: agonists serotenergic, DOI; anaesthetics volatile, halothane; analeptics, caffeine; complications, malignant hyperthermia; complications, serotonin syndrome


    Introduction
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Various studies indicate a potential influence of serotoninergic mechanisms in the development of malignant hyperthermia (MH).14 Following administration of the hallucinogenic serotonin-2 5HT2A receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) in conscious MH-susceptible (MHS) pigs, behavioural alterations, muscle rigidity, hyperthermia and metabolic alterations were observed, which were interpreted as MH.5 It is not known, however, whether these alterations were mediated by specific effects on the skeletal muscle cell, resulting in an MH crisis, or resulted from a central overstimulation eliciting a serotonin syndrome and/or from an hallucinogen-induced psychosis starting a porcine stress syndrome.

This study was designed to identify the in vivo effects of DOI on anaesthetized MHS and MH normal (MHN) pigs. In this experimental setting, a hallucinogen-induced stress reaction is excluded by anaesthesia to enable differentiation between MH and serotonin syndrome.


    Methods and results
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
After approval of the local animal research committee, five MHN (German Landrace pigs, aged 4–6 months, weighing 39–48 kg) and five MHS pigs (Pietrain pigs, aged 4–5 months, weighing 26–33 kg) were investigated. The pigs were premedicated with azaperone 4 mg kg–1 i.m. and metomidate 10 mg kg–1 intraperitoneally. General anaesthesia was induced with etomidate 0.3 mg kg–1 and fentanyl 10 µg kg–1 and maintained with etomidate 2.5 mg kg–1 h–1, fentanyl 50 µg kg–1 h–1 and nitrous oxide 66% in oxygen. After tracheal intubation, the lungs were ventilated mechanically. Neuromuscular blocking agents were not administered. A central venous catheter and an arterial cannula were placed. Radiant heat and warming blankets guaranteed normothermia during the baseline period. Rectal temperature was measured continuously. A blood gas analyser (ABL625, Radiometer, Denmark) was used to monitor PaCO2 and pH. Creatine kinase concentrations in venous plasma were measured using standard laboratory methods.

Mechanical ventilation was adjusted to maintain PaCO2 at 40 (SD 2) mm Hg until the experiment started. Once a steady state was achieved for 30 min, baseline values for all variables were recorded. Three doses of DOI 1 mg kg–1 i.v. were administered at 40-min intervals. Variables were measured every 10 min. After completion of all tests the animals were killed by i.v. magnesium chloride.

Statistical analysis was performed using SPSS® 10.0 (SPSS Inc., USA). Data are presented as medians and ranges. As an overall non-parametric ANOVA, the Friedman test for the analysis of intragroup variations was used. In cases of significance, we compared the baseline values with each time point using the Wilcoxon test. The intergroup analysis was performed with the non-parametric Mann–Whitney U-test. P<0.05 indicated statistical significance.

The median body temperature in MHS pigs before the first dose of DOI was 36.6 (range 35.8–38.5) °C. The temperature increased to 37.7 (36.9–39.6) °C 40 min after the first dose of DOI, 38.9 (38.3–42.1) °C 40 min after the second dose and 41.5 (40.2–41.9) °C 40 min after the last dose. MHN animals had a baseline body temperature of 37.8 (35.7–39.1) °C. The temperature increased to 38.7 (36.5–39.7) °C 40 min after the first dose of DOI, 39.6 (37.6–40.7) °C 40 min after the second dose and 40.3 (38.1–41.5) °C 40 min after the last dose. Statistical analysis showed a significant temperature increase in the MHS and MHN groups, but no significant intergroup variations.

PaCO2 increased significantly in both groups (Fig. 1) but there was a significant difference between the two groups only after the third dose of DOI.



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Fig 1 Effects of cumulative i.v. administration of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) 1 mg kg–1 on arterial carbon dioxide pressure (PaCO2) of malignant hyperthermia susceptible (n=5) and normal pigs (n=5). Data are medians and ranges. *Significant intragroup differences compared with baseline value; {dagger}significant difference between the MHS and MHN groups (P<0.05).

 
The first dose of DOI induced a significant decrease in pH from 7.46 (7.42–7.47) to 7.30 (7.26–7.38) in the MHS group. The second dose decreased pH to 7.24 (7.05–7.32) and the third to 7.13 (6.84–7.17). The pH values decreased significantly after the first dose in the MHN pigs from 7.49 (7.41–7.56) to 7.35 (7.31–7.44), but remained virtually unchanged for the rest of the experiment. The intergroup comparisons showed a significant difference in pH after 120 min only.

In the MHS pigs, heart rate rose significantly from the baseline value of 60 (55–94) beats min–1 to 66 (61–91) beats min–1 after the first DOI dose, 85 (65–205) beats min–1 after the second dose and 115 (87–174) beats min–1 after the third dose. In contrast, no significant changes in heart rate occurred in MHN pigs. After 120 min, however, a significant difference in heart rate was found between the groups.

Non-significant but higher baseline creatine kinase concentrations were measured in MHS pigs (median 7.7 u litre–1, range 5.5–18.9 u litre–1) compared with MHN pigs (4.8 u litre–1, 4.2–8.1 u litre–1). In both groups, the cumulative challenge of DOI exerted only slight effects on creatine kinase activity. Both MHS and MHN pigs developed skeletal muscle fasciculations. Muscle rigidity was not observed, however.

All MHS pigs fulfilled the MH criteria during the experiment. Body temperature increased by more than 2 °C in all MHN pigs and PaCO2 exceeded 70 mm Hg in two MHN animals. Thus, two MHN pigs fulfilled the criteria of MH.


    Comment
 Top
 Abstract
 Introduction
 Methods and results
 Comment
 References
 
Löscher and colleagues5 studied the in vivo effects of the 5HT2A receptor agonist DOI in conscious MHS pigs. In addition to altered behaviour, typical MH signs such as muscle rigidity and hyperthermia and metabolic changes such as elevated PaCO2, lactate and creatinine kinase concentrations and lowered pH were observed in MHS but not in MHN pigs. Thus, DOI was interpreted as an MH triggering agent. Three underlying pathways may be responsible for the MH-like signs and symptoms after the administration of 5HT2A agonists in conscious pigs. Firstly, direct substance-specific effects on peripheral sites, such as an activation of the skeletal muscle cell via 5HT2A receptors resulting in an MH crisis could be responsible. A typical fulminant MH crisis is characterized by generalized fasciculations in MHS pigs.68 Five min after the administration of trigger agents, hypercapnia, hypoxaemia, increased lactate concentrations and lowered pH are found. Cardiovascular responses include initial hypotension, tachycardia, increased cardiac output and arrhythmias. As a late sign of MH, a significant increase in body temperature is not found until 10 min after exposure to a trigger substance.8 Secondly, a porcine stress syndrome could be responsible. This syndrome describes MHS animals which are adversely affected by stress induced by trucking, fighting, coitus and crowding.9 In this context, the hallucinogen-induced psychosis in conscious pigs might lead to a central stress induction, which secondarily results in the MH-like changes in skeletal muscle metabolism. The third hypothesis emphasizes a 5HT2A-specific central agonist action, like the serotonin syndrome.10

Our in vivo experiments did not show an unequivocal difference in the defined MH criteria in the study groups. Fasciculations and comparable increases in temperature, PaCO2 and a decline in pH were monitored after the first and second challenge of DOI in both groups of pigs. However, differences in heart rate, pH and PaCO2 between MHS and MHN did not occur until the third dose of DOI.

The predictive value of our study may be reduced because the two groups of pigs had significantly different weights. (There was no difference in age.) The weight difference could possibly explain the lower resting temperature in MHS compared with MHN pigs. In addition, a lower body temperature might be an MH-protective factor in pigs.

The results of the present study are in contrast to the typical course of MH in susceptible swine. Furthermore, the well-known trigger substances cannot induce MH in normal animals. It is notable that Löscher and colleagues5 described MH crises in conscious pigs at a lower concentration of DOI (0.8 mg kg–1). Thus, it is tempting to speculate that different mechanisms are responsible for the induction of MH by 5HT2A agonists and known MH trigger agents. The prolonged onset of clinical signs and symptoms, with similarity in the course of temperature, PaCO2 and pH in the two groups of pigs is in accordance with a serotonin syndrome.10 Consequently, DOI does not seem to be a direct MH triggering agent in vivo in swine. However, 5HT2A receptor agonists might secondarily trigger a porcine stress syndrome in conscious swine by inducing central psychotic alterations, which could be prevented by anaesthesia.


    Acknowledgements
 
We are grateful to Mr Agurski of Radiometer (Denmark) for providing the blood gas analyser ABL625 and to Dr Dimigen (Head of the Department of Veterinary Medicine of the University Hospital of Hamburg-Eppendorf) and his team for providing excellent research conditions.


    References
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 Introduction
 Methods and results
 Comment
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10 Wappler F, Fiege M, Steinfath M. The role of the serotonin (5-HT) system in malignant hyperthermia. In: Schulte am Esch J, Scholz J, Wappler F, eds. Malignant Hyperthermia. Lengerich: Pabst, 2000; 176–89