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Revista Brasileira de Anestesiologia

Print version ISSN 0034-7094On-line version ISSN 1806-907X

Rev. Bras. Anestesiol. vol.67 no.1 Campinas Jan./Feb. 2017

https://doi.org/10.1016/j.bjane.2015.02.010 

Scientific Articles

The effects of intra-cerebroventricular administered rocuronium on the central nervous system of rats and determination of its epileptic seizure-inducing dose

Mehmet Baykala 

Necati Gökmena 

Alper Doğana 

Serhat Erbayraktarb 

Osman Yılmazc 

Elvan Ocmena 

Hale Aksu Erdosta  * 

Atalay Arkana 

aDokuz Eylul University, Department of Anesthesiology, Izmir, Turkey

bDokuz Eylul University, Department of Neurosurgery, Izmir, Turkey

cDokuz Eylul University, Department of Animal Research Center, Izmir, Turkey


Abstract

Background:

The aim of this study was to investigate the effects of intracerebroventricularly administered rocuronium bromide on the central nervous system, determine the seizure threshold dose of rocuronium bromide in rats, and investigate the effects of rocuronium on the central nervous system at 1/5, 1/10, and 1/100 dilutions of the determined seizure threshold dose.

Methods:

A permanent cannula was placed in the lateral cerebral ventricle of the animals. The study was designed in two phases. In the first phase, the seizure threshold dose of rocuronium bromide was determined. In the second phase, Group R 1/5 (n = 6), Group 1/10 (n = 6), and Group 1/100 (n = 6) were formed using doses of 1/5, 1/10, and 1/100, respectively, of the obtained rocuronium bromide seizure threshold dose.

Results:

The rocuronium bromide seizure threshold value was found to be 0.056 ± 0.009 µmoL. The seizure threshold, as a function of the body weight of rats, was calculated as 0.286 µmoL/kg-1. A dose of 1/5 of the seizure threshold dose primarily caused splayed limbs, posturing, and tremors of the entire body, whereas the dose of 1/10 of the seizure threshold dose caused agitation and shivering. A dose of 1/100 of the seizure threshold dose was associated with decreased locomotor activity.

Conclusions:

This study showed that rocuronium bromide has dose-related deleterious effects on the central nervous system and can produce dose-dependent excitatory effects and seizures.

KEYWORDS Rocuronium; Seizure; Central nervous system; Rat

Resumo

Justificativa:

O objetivo deste estudo foi investigar os efeitos do brometo de rocurônio administrado intracerebroventricularmente sobre o sistema nervoso central, determinar a dose do limiar convulsivo de rocurônio em ratos e investigar os efeitos de rocurônio no sistema nervoso central em diluições de 1/5, 1/10 e 1/100 da dose do limiar convulsivo determinada.

Métodos:

Uma cânula permanente foi colocada no ventrículo lateral do cérebro dos animais. O estudo foi projetado em duas fases. Na primeira, a dose do limiar convulsivo do brometo de rocurônio foi determinada. Na segunda, o Grupo R 1/5 (n = 6), o Grupo 1/10 (n = 6) e Grupo 1/100 (n = 6) foram formados com doses de 1/5, 1/10 e 1/100, respectivamente, da dose do limiar convulsivo de brometo de rocurônio obtida.

Resultados:

Descobrimos que o valor do limiar convulsivo de brometo de rocurônio é 0,056 ± 0,009 µmoL. O limiar convulsivo, como uma função do peso corporal dos ratos, foi calculado como 0,286 µmoL/kg-1. Uma dose de 1/5 da dose do limiar convulsivo causou principalmente abertura postural dos membros e tremores em todo o corpo, enquanto uma dose de 1/10 da dose do limiar convulsivo causou agitação e tremores. Uma dose de 1/100 da dose do limiar convulsivo foi associada à diminuição da atividade locomotora.

Conclusões:

Este estudo mostrou que o brometo de rocurônio tem efeitos deletérios relacionados com a dose sobre o sistema nervoso central e pode produzir efeitos excitatórios dependentes da dose e convulsões.

PALAVRAS-CHAVE Rocurônio; Convulsão; Sistema nervoso central; Rato

Introduction

In cases in which neuromuscular blocking drugs have been administered accidentally into the cerebrospinal fluid (CSF),1 myotonia, autonomic changes, and convulsions have occurred.2 Prolonged administration of rocuronium in critical care patients has resulted in the drug entering the CSF.3 Also in patients with impaired function of the blood brain barrier these molecules may reach the CSF. Tassonyi et al.4 has reported that some neuromusculer agents like atracurium and its metabolite laudanosine present in the CSF, vecuronium could not be detected in CSF in the subarachnoid hemorrhage. And also atracurium persisted in the CSF for several hours after the end of the infusion. Although it is a non-depolarizing, ionized medication with relatively low lipophilicity, it is known that rocuronium can permeate the CSF after intravenous injection.5 Accidental administration of rocuronium into the central nervous system (CNS) during regional anesthesia has been reported.6 There are limited data regarding the effects of these drugs on the CNS.

The aim of this study was to investigate the effects of intracerebroventricularly administered rocuronium bromide on the CNS, determine the seizure threshold dose of rocuronium bromide in rats, and investigate the effects of rocuronium on the CNS at 1/5, 1/10, and 1/100 dilutions of the seizure threshold dose.

Methods

The study protocol was approved by the Animal Research Committee of Dokuz Eylul University. Thirty-six female Wistar albino rats that were bred in the Experimental Research Laboratory of Dokuz Eylul University were used in the study. The rats weighed 180-260 g and were 87% homogeneous, were aged 18-24 months, and exhibited normal activity. The animals were kept under standard laboratory conditions for one week before the experiments began (12 h light: 12 h light, 20-22 ºC room temperature) to adapt to the environment. They had free access to water and pellet food, and the study was conducted in a noise-attenuated environment and at the same time each day.

Surgery

Anesthesia was induced by intraperitoneal injection of 50 mg/kg-1 sodium thiopental (Pental® Sodyum I.E. Ulugay Ilaç San. TAS, Istanbul, Turkey) while maintaining spontaneous respiration and the blink reflex. The rats were placed in the prone position, and a 4 cm × 2 cm area between the ears and extending toward the nasus was shaved. The area was cleaned with povidon iodine, 1 mL of sterile 1% lidocaine (Aritmal® 2% amp, Biosel Ilaç Sanayi ve Ticaret A.S, Istanbul, Turkey) was infiltrated under the scalp, and a midline surgical incision was made. The head of the animal was placed in a stereotaxic frame where the head was secured in a flexed-forward position. The skin was laterally reflected, and the exposed cranium was gently blunt-dissected with the bregma exposed. A 2 mm hole for the intracerebroventricular rocuronium bromide injection was bored at the stereotaxic coordinates, determined according to the atlas of Paxinos and Watson,7 to access the lateral ventricle. The cannula used for the intracerebroventricular procedures (C311/G, 20G, Plastics One Inc., VA, USA) and a suitable stylet (C311/DC, Plastics One Inc., VA, USA) to prevent tissue and/or foreign bodies from moving into the cannula were placed. The placement of the cannula in the lateral ventricle was confirmed by CSF drainage through the cannula. The edges of the incision were approximated, sutured with 2/0 silk thread, and cleaned with 10% povidon iodine. These procedures were conducted under sterile conditions. Forty-eight hours were allowed for the usual activity of the rats to be restored after this intervention. The purpose of placing a permanent cannula was to enable ICV drug administration while the rats were awake and mobile.

Preparation of the drug

A stock solution with 0.016 µmoL rocuronium bromide (Esmeron®, Organon Corp, Oss, Holland) in 10 µL was used to determine the seizure threshold dose of rocuronium bromide.

Following the determination of the seizure threshold dose of rocuronium, the 1/5, 1/10, and 1/100 dilutions in 10 µL Ringer's lactate solution (Ringer lactate, Biosel Ilac San. TAS, Istanbul, Turkey) were prepared.

The pH values of the rocuronium bromide, Ringer's lactate, and rocuronium bromide-Ringer's lactate solutions at 23 ºC were measured with a pH meter (InoLab® 720, WTW Wissenschaftlich-Technische Werkstätten GmbH, Munich, Germany).

Experimental protocol

Determination of the seizure threshold dose of rocuronium bromide (Pilot study): To determine the seizure threshold dose of rocuronium bromide, a total of 12 rats were randomized into 2 groups.

Group rocuronium (n = 6): To determine the seizure threshold dose, a vinyl-coated polyethylene adapter was attached to the ICV cannula and a Hamilton micro-syringe was attached to this adapter (Hamilton® 710SNR 100 µL Syringe Hamilton 710 series syringe, NV, USA). Rocuronium bromide (0.016 µmoL × 10 µL-1, total 0.08 µmoL) was injected via the Hamilton syringe in divided doses of 5 µL. Each dose was administered consecutively over 60 s, during which time the effects of the dose on the rats were observed. The total dose required to induce a tonic-clonic seizure was recorded.

Group control (n = 6): A total of 50 µL of Ringer's lactate solution, in divided doses of 5 µL, was administered via the ICV cannula, as explained above. The rats that did not have seizures were sacrificed after a 6 h observation period.

Dose-response study groups (experimental study): After the determination of the seizure threshold dose of rocuronium bromide, the rats were randomized into 4 groups for the dose-response study.

Group 1 (Group C) (n = 6): Ten microliters of a mixture of Ringer's lactate solution and acetic acid with a pH identical to that of the rocuronium solution that induced seizures was administered with the Hamilton syringe over 60 s.

Group 2 (Group R 1/5) (n = 6): Using the same method as that used for Group C, 1/5 of the seizure dose of rocuronium was added to the Ringer's lactate solution to obtain a total solution volume of 10 µL and administered to the rats.

Group 3 (Group R 1/10) (n = 6): Using the same method as that used for Group C, 1/10 of the seizure dose of rocuronium was added to the Ringer's lactate solution to obtain a total solution volume of 10 µL and administered to the rats.

Group 4 (Group R 1/100) (n = 6): Using the same method as that used for Group C, 1/100 of the seizure dose of rocuronium was added to the Ringer's lactate solution to obtain a total solution volume of 10 µL and administered to the rats.

Assessment of the effects of intracerebroventricular rocuronium bromide: A five-point scale was used to assess the CNS effects of rocuronium bromide administered intracerebroventricularly.8

  • 0 = no observable effects

  • 1 = decreased locomotor activity and/or piloerection

  • 2 = agitation or shivering

  • 3 = entire body tremors, posturing, or splayed limbs

  • 4 = tonic-clonic convulsions or seizures

Termination of the study

The animals that had seizures were immediately sacrificed with an intraperitoneal injection of 120 mg/kg-1 sodium thiopental. The animals that did not have a seizure were observed for 6 h and sacrificed with 120 mg/kg-1 intraperitoneal sodium thiopental at the end of the study.

All the animals were injected postmortem with 50 µL of methylene blue via the permanent cannula into the cerebral ventricle, and the brain was bisected along the longitudinal fissure to examine whether the dye was uniformly distributed within the ventricle.

Statistical analysis

The statistical analyses were performed using the SPSS for Windows v. 11.0 statistical package. The results are expressed as the mean ± standard deviation. The Kruskal-Wallis test, followed by the Mann-Whitney U and Fisher's exact tests, was used for the between-group comparisons. The within-group comparisons were conducted using Friedman and Wilcoxon tests, and p < 0.05 was considered statistically significant.

Results

Determination of seizure threshold (pilot study)

The rats in the control group, which received a total of 50 µL of Ringer's lactate solution (pH = 5.2) in the lateral cerebral ventricles, did not exhibit any behavioral alterations.

In the rocuronium group, the seizure threshold for ICV rocuronium bromide (pH = 3.6) was found to be 0.056 ± 0.009 µmoL. The volume necessary to induce a seizure was found to be 35.0 ± 5.48 µL. The seizure threshold, as a function of the body weight of rats, was calculated as 0.286 µmoL/kg-1 (Table 1).

Table 1 Rocuronium bromide doses and volumes. 

Weight
(g)
Dose
(µmoL)
Dose
(µmoL/kg)
Volume
(µL)
201 0.064 0.326 40
198 0.064 0.331 40
212 0.064 0.308 40
206 0.048 0.239 30
198 0.048 0.248 30
184 0.048 0.267 30

The seizure threshold, as a function of the body weight of rats, was calculated as 0.286 µmoL/kg.

Evaluation of the dose-response results study (experimental study)

The pH values of the Group C, Group R 1/5, Group R 1/10, and Group R1/100 were determined to be 3.6, 3.9, 4.1, and 4.8, respectively.

In this study, we found that 1/5 of the seizure threshold dose typically caused splayed limbs, posturing, and entire body tremors, whereas 1/10 of the seizure threshold dose caused agitation and shivering (Tables 2 and 3).

Table 2 CNS effects of rocuronium at 1/5 of the seizure threshold dose. 

Weight (g) CNS effect Score
189 Seizure 4
266 Extremity gets a posture 3
232 Extremity gets a posture 3
238 Extremity gets a posture 3
199 Extremity gets a posture 3
184 Seizure 4

Table 3 CNS effects of rocuronium at 1/10 of the seizure threshold dose. 

Weight (g) CNS effect Score
194 Shivering 2
180 Extremity posture 2
182 Extremity posture 2
217 Agitation 3
187 Agitation 3
189 Shivering 2
One-hundredth of the seizure threshold dose was associated with decreased locomotor activity (Table 4). The effects observed at these doses gradually improved over a 1-h period and were then maintained as reduced locomotor activity. The animals were observed for a total of 6 h, during the time when feeding and motor behaviors became comparable to those of the control group.

Table 4 CNS effects of rocuronium at 1/100 of the seizure threshold dose. 

Weight (g) CNS effect Score
218 Decreased locomotor activity 2
196 Decreased locomotor activity 1
192 Decreased locomotor activity 3
202 Decreased locomotor activity 1
186 Shivering 2
191 Decreased locomotor activity 1

Discussion

In this study, we found that rocuronium administered into the central nervous system of rats through the cerebral ventricles caused seizures when the rats were not under anesthesia and that the seizure threshold dose of rocuronium was 0.286 µmoL/kg-1. We determined during the dose-response study that 1/5 and 1/10 of the seizure threshold dose of rocuronium yielded excitatory responses, while 1/100 of the dose resulted in decreased locomotor activity.

In the pilot study, behavioral changes were not observed in the rats when we administered 100 µL of Ringer's lactate solution at a rate of 5 µL/min-1. We administered a maximum of 40 µL while determining the epileptic seizure threshold dose. We do not hypothesize that the volume of the drug is responsible for the CNS changes observed in rats. In this study, we determined that the pH of the drug administered via the ICV cannula was not a contributing factor to the induction of seizures because only reduced locomotor activity was observed in Group 1.

In a similar study, Szenohradszky and colleagues administered atracurium, pancuronium, and vecuronium into the CNS and observed the side effects, finding the epileptic seizure potencies of atracurium > pancuronium > vecuronium (0.12, 0.26, and 0.46 µmoL/kg-1, respectively).8 In the present study, the potency of rocuronium necessary to cause seizures was found to be near that of pancuronium.

In cases in which neuromuscular blocking drugs have been administered accidentally into the CSF,1 the diffusion of rocuronium into the neuronal tissue from cerebrospinal fluid is likely a function of molecular weight, lipid solubility, and time.9 Based on the physical properties of rocuronium, it is likely to remain in the cerebrospinal fluid after injection, with rapid penetration into neuronal tissues being unlikely.9 Drugs in the cerebrospinal fluid can be redistributed into the peripheral circulation by transport across the blood-brain barrier. An ICV infusion may, in some instances, mimic a slow intravenous infusion.10 We did not observe such an incidence at any stage of the study, which may have occurred because the dose of rocuronium that entered the systemic circulation was not sufficient to reach the concentration necessary to reach the neuromuscular junction and cause muscle relaxation.

ICV-administered drugs may exert receptor-mediated effects at or near the brain-cerebrospinal fluid interface. Rocuronium is a quaternary ammonium compound. The positive charge of this molecule mimics that of the quaternary nitrogen atom of Acetylcholine (Ach). This atom is the major contributor to the effects of rocuronium on the neuronal nAchr.11 There are insufficient data on the kinetics of the distribution of muscle relaxants in the CSF when administered into the cerebral ventricles,8,12 and it is not known in which part of the brain the ACh receptors mediate the inhibitory and excitatory effects observed with rocuronium.

As with all muscle relaxants, the site of action of rocuronium is the postsynaptic ACh receptor at the neuromuscular junction. In an experimental study, Jonsson et al.13 demonstrated that rocuronium inhibited muscular and neuronal nAchr. Neuronal nAchr is irreversibly inhibited by rocuronium at the micro-molar level in a concentration-dependent manner. These findings suggest that rocuronium exerts only inhibitory effects on the ACh receptors in the neuromuscular junction, while it exerts inhibitory and excitatory effects on the nicotinic ACh receptors in the CNS.

Fuchs-Buder et al.6 and Tassonyi et al.14 reported that neuromuscular blocking agents may affect the CNS, as evidenced by the induction of apnea.

Shao et al.15 demonstrated that the cholinergic nervous system is important in the regulation of respiratory patterns, and inhibition of nAch may cause central depression of respiration. In our study, we observed no sign of depression of respiration or apnea.

The behavioral changes in rats manifested as decreased locomotor activity after 1 h and recovery of normal movement and feeding behavior after 6 h were attributed to rapid CSF clearance, which is 2.83 µL/min-1 in rats.16 Because the CSF volume in rats is 500 µL, the total CSF clearance time is 176 min.4,8 Meulemans et al.17 reported that 45% of the CSF of a 290 g rat was cleared in 1 h, which is in accordance with our findings.

The exact mechanisms of action of muscle relaxants on the central nervous system have not been established. This study showed that rocuronium is effective on the CNS and has dose-related deleterious effects. Based on the results of this study, it is not possible to establish a relationship between the effects of rocuronium in humans and rats. The amount of rocuronium accumulation in the cerebrospinal fluid in patients receiving long-term rocuronium infusion is unknown.

It should be considered that CNS effects can be observed if rocuronium is infused over longer periods of time and in cases in which the blood-brain barrier is compromised or rocuronium is administered inadvertently during a regional block.

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Received: December 12, 2014; Accepted: February 23, 2015

* Corresponding author. E-mail:hale.erdost@deu.edu.tr (H.A. Erdost).

Conflicts of interest

The authors declare no conflicts of interest.

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