CHANGES IN BIOMECHANICAL PARAMETERS DURING HEART PERFUSION AND AFTER MIDAZOLAM PRE-MEDICATION – EXPERIMENTAL PILOT STUDY

Background: Midazolam is a frequently used benzodiazepine in anaesthesiology and intensive care. Aim: The aim of pilot study was to monitor its eff ect during heart perfusion in the laboratory rat. Methods: The same groups of animals (n = 10). The 1 group was treated with midazolam in a dose of 0.5mg/kg i.p. The 2 group was a placebo. After i.p. administration of heparine injection of 500 IU dose, the hearts were excised and perfused (modifi ed Langendorf’s method). Working schedule: stabilization/ischaemia/reperfusion proceed at intervals of 20/30/60 min. Monitored parameters in isolated heart: left ventricle pressure (LVP), end-diastolic pressure (LVEDP), contractility (+dP/dtmax). Results: The treated hearts showed improved postischemic recovery, reaching LVP values of 92 ± 6 % at the end of the reperfusion, placebo only 61 ± 7 %. In placebo hearts LVEDP rose from 10.0 ± 0.5 mmHg to 43 ± 4 mmHg after, in treated animals only about 25 mmHg. The treated hearts improved +dP/dtmax recovery during reperfusion to 91 ± 8 %. These values were signifi cantly greater than those obtained from the placebo hearts. Conclusions: Positive changes in monitored parameters were found in this experimental pilot study. We conclude that the administration of midazolam in laboratory rats has a cardioprotective potential against ischemia-reperfusion induced injury.


INTRODUCTION
Midazolam is a benzodiazepine derivative.It has powerful anxiolytic, amnestic, hypnotic, anticonvulsant, skeletal muscle relaxant and sedative properties.It is considered a fast-acting benzodiazepine, with a short elimination half-life.It is therefore a very useful drug to use for short minor procedures such as dental extraction.Midazolam was fi rst synthesized in 1976 by Fryer and Walser 1 .
Like other benzodiazepines, midazolam acts on benzodiazepine receptors to enhance the binding of GABA to the GABA A receptor which results in inhibitory eff ects on the central nervous system 2 .
Midazolam is indicated for the acute management of aggressive or delirious patients and also is sometimes used for the acute management of seizures such as status epilepticus.Long term use for the management of epilepsy is not recommended however, due to the signifi cant risk of tolerance which renders midazolam and other benzodiazepines ineff ective and as well the signifi cant side-eff ect of sedation 3 .In mice given chronic midazolam a slowly evolving tolerance developed to the anticonvulsant properties of midazolam over 15 days, although some anticonvulsant eff ects were still apparent after 15 days of continued administration 4 .
Midazolam is occasionally used as a hypnotic, especially in hospitals.Like other benzodiazepines, it produces a decrease in delta activity, though the eff ect of benzodiazepines on delta may not be mediated via benzodiazepine receptors.Delta activity is an indicator of depth of sleep within non-REM sleep; it is thought to refl ect sleep quality, with lower levels of delta sleep refl ecting poorer sleep.Thus midazolam and other benzodiazepines cause deterioration in sleep quality.Cyproheptadine may be superior to nitrazepam in the treatment of insomnia as it enhances sleep quality based on EEG studies 5 .
Midazolam is frequently used benzodiazepine in anaesthe siology and intensive care, too.
The aim of the study was to monitor the eff ects of midazolam during heart perfusion of laboratory rat.

METHODS
The study and its experimental protocol were approved and monitored by the Ethics Committee of Palacký University.The state of health of all animals was inspected regularly several times a day both during the acclimation of the animals and in the course of the whole experiment performed by the working group whose members are holders of the Eligibility Certifi cate issued by the Central Commission for Animal Protection pursuant to Section 17 of the Czech National Council Act No 246/1992 Coll. on animal protection against maltreatment.
This study was performed on 20 male Wistar SPF (AnLab, Germany) laboratory rats of the same age (6 months) and comparable weight (345 ± 15 gr).The L. Bartosikova, J. Necas, T. Bartosik, P. Frana, M. Pavlik animals were housed in a standard controlled temperature, fed the standard diet for small laboratory animals, and given water ad libitum.After a recovery period, the animals were divided randomly into 2 groups (n = 10).
The fi rst group -treated group -received midazolam in a single dose 0.5 mg/kg by i.p. injection.The second group -the placebo group -received only normal saline solution also by i.p. injection.
The rats were anesthetized with an i.p. injection of anaesthetical mixture (2% Rometar 0.5 ml + 1% Narkamon 10 ml, dose 0.5 ml solution/100 g body weight).After the i.p. heparine injection of 500 IU dose, the hearts were excised and perfused.In all experiments, the modifi ed Langendorff method and the universal apparatus Hugo Sachs Electronic UP 100 (Germany HSE) were used.Schedule: stabilization/ischaemia/reperfusion proceeded at intervals of 20/30/60 min.Biomechanical parameters from isolated heart: left ventricle pressure (LVP), enddiastolic pressure (LVEDP), contractility (dP/dt max ) were measured using a ball fi lled with liquid (8-12 mmHg), inserted through the left atrium in the left ventricle connected to the analog convertor (Isotec HSE, DIF modul HSE) 6 .

DISCUSSION
A variety of laboratory and clinical studies clearly indicate that exposure to anaesthetic agents can lead to a marked protection of the myocardium against ischaemia-reperfusiuon injury.Several changes in the protein structure of the myocardium that may mediate this cardioprotection have been indentifi ed.Ischaemia-reperfusion of the heart occurs in a variety of clinical situations including transplantations, coronary artery bypass grafting and vascular surgery.Ischaemia may also occur during stressful anaesthetic induction.Early restoration of arterial blood fl ow and measures to improve the ischaemic tolerance of the tissue are the main therapeutic options (i.e.cardioplegia and betablockers).There is increasing evidence that anaesthetic agents interact with the mechanisms of ischaemia-reperfusion injury and protect the myocardium by a preconditioning and postconditioning mechanism [7][8][9][10] .
In 1996, specifi c protection against myocardial reperfusion injury by halothane was described 11 .While all previous studies had been unable to discriminate between anti-ischemic eff ects and eff ects against reperfusion injury, this study demonstrated for the fi rst time that modifi cation of the reperfusion conditions by administration of a common volatile anaesthetic specifi cally reduced reperfusion damage.A similar cardioprotective eff ect was confi rmed for enfl urane, isofl urane, sevofl urane and desfl urane and the noble gas xenon under a variety of ex- In hearts from placebo animals, LVP recovered up to 61 ± 7 % of pre-ischemic values at the end of the reperfusion.In the midazolam pretreated animals, the hearts showed signifi cantly better post-ischemic recovery, reaching LVP values of 92 ± 6 % at the end of the reperfusion.In hearts from placebo animals LVEDP rose from 10.0 ± 0.5 to 43 ± 4 mmHg after 60 min of reperfusion.This increase was diminished in the hearts from the midazolam pretreated animals at the end of reperfusion.The pretreatment with midazolam improved +dP/ dt max recovery during reperfusion to 91 ± 8 % after 60 min of reperfusion.These values were significantly greater than those obtained from placebo hearts.Changes in biomechanical parameters during heart perfusion and after midazolam pre-medication -experimental pilot study perimental conditions in vitro and in vivo; cardioprotection against reperfusion damage was also maintained when the heart was already protected against ischaemic damage by cardioplegic solutions 12,13 .
The benzodiazepines have hardly been investigated for myocardial protection.Whether these intravenous anesthetics interfere with neutrophil-endothelium interaction, Ca 2+ infl ux and free radical production still needs to be elucidated [14][15][16][17][18] .It is known, that peripheral benzodiazepine receptors are present in peripheral tissues such as adrenals, kidney and heart, as well as in the brain 19,20 .The peripheral benzodiazepine receptor is diff erent from the central benzodiazepine receptor, which is coupled to GABA receptors and responsible to the classical sedative, anxiolytic and anticonvulsant eff ect 21,22 .Peripheral benzodiazepine receptor is a 169-amino acid protein with fi ve trans-membrane domains associated with the mitochondrial outer membrane 23,24 which has been suggested to be involved in the control of several mitochondrial functions including the respiratory chain and ion channel activities, in the regulation of apoptosis, which occurs during cardiac injury 25,26 and in the modulation of immune functions, steroidogenesis and neurodegenerative process 24,27 .
Midazolam is extensively used as hypnotic and anesthetic in clinical situations 28 .Midazolam has been reported to impair memory retention at sedative doses in human studies, and retrograde amnesia after recovery from anesthesia with anesthetic doses is often observed in humans 29 .Midazolam is used in pediatric practice too 30 .Midazolam benzodiazepines have not been investigated extensively in the adult population and no references were found in the literature to their myocardial eff ects in pediatric cardiac surgical patients.
In isolated perfused guinea pig hearts subjected to ischaemia midazolam reduced neutrophil adhesion to nonischaemic control levels.Adhesion of polymorphonuclear neutrophils to the coronary endothelium is a crucial step in the development of ischemic myocardial injury 14 .
Midazolam may interfere with Ca 2+ infl ux and free radical production but the data are contradictory in this respect 31 .In the study of Papaioannou et al. 32 some interesting anesthetic eff ects on mitochondrial and sarcolemmal K + ATP channels were described.This study has showed that barbiturates inhibit mitochondrial K + ATP channels while propofol, etomidate and midazolam have no eff ect on the above proteins and fi nal myocyte survival in rat models.The eff ect of midazolam on sarcolemal K + ATP channels activity still needs to be elucidated 33 .
An important regulator of cardiac function is adenosine.Its cardiac eff ects are predominately mediated by A 1 and A 2A receptors.Activation of A 1 receptors reduces oxygen demand by causing a negative chronotropic and dromotropic eff ect, and by inhibiting catechoamine-stimulated increases in ventricular contractility, whereas activation of A 2A receptors increases oxygen supply by causing coronary vasodilatation.The physiologic properties of adenosine convey cardioprotection during myocardial ischemia 34 .Some drugs that weakly inhibited adenosine metabolism reduced the incidence of early cardiac death and myocardial infarction after coronary artery bypass grafting 35 .Benzodiazepines reduce adenosine degradation by weakly inhibiting the nucleoside transporter, the major mechanism whereby the eff ect of adenosine is terminated through reuptake into cells and reincorporation into the intracellular nucleoside pool.In the functional studies, benzodiazepines augmented the negative inotropic eff ect of exogenous adenosine in guinea pig atrial strips 36,37 .Binding and functional studies have been conducted in cardiac tissue for the most commonly used benzodiazepine in the perioperative setting, midazolam.Seubert et al. 38 showed that midazolam selectively potentiates the A 2A receptor-mediated eff ects of adenosine.
From the results of our experiment it can be deduced that the administration of midazolam in the laboratory rats has cardioprotective potential against ischemia-reperfusion induced injury in rat hearts.This eff ect is demonstrated in the functional parameters of hearts, LVP, LVEDP and +dP/dt max .
From recent studies there is increasing evidence that cardioprotection by anaesthetic agents can be elicited in the clinical setting and may add to other organ protection strategies.Thus, it is conceivable that the choice of anaesthetic drug may have an impact on patient outcome in ischemia-reperfusion situations.However, this still has to be confi rmed by large studies that examine defi nitive outcome parameters.

Fig. 1 .
Fig. 1.Left ventricle pressure.In hearts from placebo animals, LVP recovered up to 61 ± 7 % of pre-ischemic values at the end of the reperfusion.In the midazolam pretreated animals, the hearts showed signifi cantly better post-ischemic recovery, reaching LVP values of 92 ± 6 % at the end of the reperfusion.