Possible role of high calcium concentrations in rat neocortical neurons in inducing hyper excitatory behavior during emergence from sevoflurane: a proposed pathophysiology

Abstract

Sevoflurane has been shown to increase the incidence of emergence delirium in children; however, the mechanism remains unclear. Sevoflurane increases cytoplasmic calcium concentration which in turn may play a role in emergence delirium. This study aimed to investigate the level of intracellular calcium in rats experiencing hyperexcitatory behavior after exposure to sevoflurane, as well as the role of magnesium in preventing this phenomenon. After ethical approval, 2-5-week-old Sprague-Dawley rats (n = 34) were insufflated with sevoflurane in a modified anesthesia chamber. One group received magnesium sulphate intraperitoneally. After termination of sevoflurane exposure, the occurrence of hyperexcitation was observed. Brain tissue samples from the rats were studied for intracellular calcium levels under a two-channel laser scanning confocal microscope and were quantitatively calculated using ratiometric calculation. The presence of inflammation or oxidative stress reaction was assessed using nuclear factor κB and malondialdehyde. The incidence of hyperexcitatory behavior post sevoflurane exposure was 9 in 16 rats in the observation group and none in the magnesium group. Tests for inflammation and oxidative stress were within normal limits in both groups. The rats showing hyperexcitation had a higher level of cytosol calcium concentration compared to the other groups. To conclude, the calcium concentration of neocortical neurons in Sprague-Dawley rats with hyperexcitatory behavior is increased after exposure to sevoflurane. Administration of magnesium sulphate can prevent the occurrence of hyperexcitation in experimental animals.

INTRODUCTION

Emergence delirium is a clinical condition marked by a variety of behavioral changes in children immediately after regaining consciousness shortly after anesthesia.1 It is known to cause serious problems, contributing to postoperative complications. The phenomenon occurs in both children and adults; however, it is more prominent in children. Emergence delirium increases the risk of patient safety incidents, increases workload, and may prolong the patient’s length of stay, thereby increasing the overall healthcare cost.2,3 The etiology of emergence delirium has yet to be understood, thus, an optimal course of action has not been determined yet.

Yasui et al.4 showed that sevoflurane provokes emergence agitation in rats, by directly activating an inward current that excites the locus coeruleus neurons. Previous studies also showed that sevoflurane has been shown to increase intracellular calcium concentration.5,6 Kindler et al.7 documented that the increase in intracellular calcium concentration by sevoflurane depended on the release of calcium from intracellular storage and was not related to extracellular calcium concentration. Exposure to sevoflurane is an external factor that induces the activation inositol 1,4,5-triphosphate (InsP3) receptors that stimulates the release of calcium from the calcium-sensitive storage of the endoplasmic reticulum (ER).8 An excess amount of intracellular calcium is thought to trigger a hyperexcitability state in neurons, as it does in the cardiac muscle, resulting in emergence delirium.

The role of magnesium in the field of anesthesia has received more attention from clinical researchers. Magnesium has been known for its effect to reduce the need for anesthesia drugs and attenuate the cardiovascular response during painful stimulation such as laryngoscopy and intubation.9 Clinically, the administration of magnesium can reduce the incidence of delirium emergence in children.10 The dynamics of intracellular calcium are thought to be related to magnesium concentration. High extracellular Mg²⁺ concentration will prevent calcium influx into the cell by inhibiting voltage-sensitive calcium channels. Magnesium ions work as non-competitive inhibitors of InsP3 bonds in the InsP3 calcium-sensitive channels.11

Several in vitro studies involving cell cultures and experimental animals have been conducted to investigate emergence delirium. However, none has been able to elucidate the pathophysiology of emergence delirium.3 This study aimed to investigate the level of intracellular calcium in young rats experiencing post-anesthesia hyper-excitatory behavior (HEB), comparable to emergence delirium, and the effect of a non-selective calcium antagonist (magnesium) in preventing this phenomenon.

MATERIALS AND METHODS

Animals

The study received ethical approval from the Ethical Committee for Research in Humans and Animals from Faculty of Medicine, Universitas Indonesia (approval number: 445/ UN2.F1/ETIK/2017) on May 15, 2017. Thirty-four Sprague-Dawley rats (Rattus norvegicus) of both sexes aged 2–5 weeks and weighing 50 to 100 g were secured from the Center for Biomedical and Health Technology, Indonesian Ministry of Health Research and Development Center (PBTDK Balitbang Ministry of Health). The animal care was performed according to the laboratory standard of care. The number of animals was calculated based on Federer’s calculation12, with six rats in each group. However, there is a lack of studies regarding the incidence of emergence delirium in rats. Hence, we based our calculation on the incidence of emergence delirium in our institution, 40%. This study randomized 32 rats from the same parent into two different groups, namely the observation (n = 16) and the magnesium groups (n =16).

Design

The experimental procedure was divided into two stages; the first was an in-vivo study to observe the incidence of post-anesthesia excitatory behavior comparable to emergence delirium in rats. The next stage was an in-vitro procedure to discover the concentration of intracellular calcium, inflammation, and oxidative stress conditions in the rats’ acute brain slices. We included 32 rats for the in vivo study and 24 rats for in vitro study (Figure 1).

Figure 1.

The process of group assignment and randomization.

Note: HEB: hyperexcitatory behavior.

In vivo study

The rats were placed in an anesthesia chamber, where anesthesia was induced with 5% sevoflurane (Abbott Laboratories London, UK, Tec3 Sevoflurane Vaporizer, scale 0–5 vol%) in 40% oxygen at the rate of 5 L/min, until the loss of righting reflexes of the subjects. The anesthesia then was maintained with 2.5% sevoflurane for 10 minutes. The level of sevoflurane in the anesthesia chamber was confirmed by an authorized calibration company. The subjects in the magnesium group were injected with magnesium sulphate (MgSO₄, 30 mg/kg) soon after anesthesia induction and the loss of righting reflex. A cut-off time of 30 minutes for behavioral observation post-sevoflurane exposure was used to determine whether the subjects showed any HEB. The animals’ hyperexcitation behaviors were scored using modified quantitative scoring by Bough et al.13 with scoring as follows; 0: no hyperexcitation changes, 1: trembling, 2: head bobbing, 3: unilateral limb clonus, 4: bilateral limb clonus, 5: rolling and 6: wandering.13,14,15 The total duration and most evident symptoms of HEB were recorded. Rats without excessive excitation behavior were used as the control group.

In vitro study

Subjects’ group was extended according to the occurrence of post-anesthesia HEB (Figure 1). The experiment was continued by decapitating the rats using the cervical dislocation technique to harvest the brain tissue, with the decapitation performed by skilled laboratory technicians. The brain slices were prepared with a modified procedure based on a study previously conducted by Lim et al.16 The brain tissue was submerged in iced cold artificial cerebrospinal fluid containing (in mM): NaCl 130, KCl 4, CaCl₂ 2.5, MgCl₂ 1,4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) 1 and glucose 11. The chilled brain tissue containing the frontal cortex was trimmed using a microtome (VT1000, Leica Biosystem, Deer Park, IL, USA) to obtain two sizes of slices – 200 μm for the confocal preparation. The slices were submerged into artificial cerebrospinal fluid without calcium and perfused with carbogen (oxygen 95% and CO₂ 5%). The slices were then bath loaded with 1 μΜ Fura Red (Fura Red dye [Fura-Red™ Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA] dissolved with dimethyl sulfoxide and pluronic acid) and incubated at 37°C for 30 minutes before they were ready for examination under a two-channel laser scanning confocal microscope (FV 1000, Olympus Lifescience, Tokyo, Japan] (Additional Table 1) connected to the Fluoview FV1000 version 1.4.1.5 program (Olympus Lifescience, Tokyo, Japan]. The intensity of the luminescence produced from the binding of free calcium and FuraRed was captured and analyzed using the ImageJ program (ver. 1.51u; National Institutes of Health, Bethesda, MD, USA).17 Ratio metric calculation was used to calculate the cytosol calcium concentration with the dissociative constant (Kd) of Fura Red AM 140 nM.18,19,20 The procedures were performed within 1 hour.

Additional Table 1.

The wavelength of two-channel laser scanning confocal microscope

Parameter	Channel 1	Channel 2	Channel 3
Dye	Fura Red (Ca Free)	TRITC	None
Emission wavelength (nm)	670	578	0
Excitation wavelength (nm)	488	543	633
Picture coloring (LUT)	Green	Red	Gray
Filter	Gausian Blur	Gausian Blur	Gausian Blur

Note: LUT: Lookup table; TRITC: tetramethylrhodamine.

The remaining brain tissue was preserved for oxidative stress and inflammation analysis. Inflammation reaction was indicated by measuring the amount of nuclear factor κB (NFκB) with the enzyme-linked immunosorbent assay method, using a special kit from MyBiosource™, San Diego, CA, USA, while oxidative stress was marked by the amount of malondialdehyde (MDA) (Bioassay Technology Laboratory, Shanghai, China) using Will’s method.21 MDA has been pointed out as the main product to evaluate lipid peroxidation.22 The values of NFκB and MDA were compared with the standard value.

All statistical analyses were done using SPSS Ver 24.0 software (IBM, Armonk, NY, USA). Numerical data distribution was tested using the Kolmogorov-Smirnov test. The comparison of cytosol calcium concentration was tested with Mann-Whitney test for data without normal distribution. A P value of < 0.05 was deemed significant. Animals that were not included in the in vitro study were kept in the cages and treated according to the standards of animal care from the laboratory animals.

RESULTS

In vivo study

The incidence of post-anesthesia HEB was observed in 9 out of 16 rats in the observation group (56%), while no such behavior was observed in the Magnesium group (Figure 1). The average induction time for all animals was 4.5 minutes. The symptoms arose within 15 minutes after sevoflurane discontinuation and the animals emerging from anesthesia. The most predominant hyperexcitation symptom observed was head bobbing followed by trembling and unilateral limb seizure. The measurement result for enzyme-linked immunosorbent assay NFkB and MDA from the subject’s brain did not show a significant increase or decrease, compared with control, in either group (Table 1).

Table 1.

The concentration of NFκB and MDA in brain tissues of rats with hyperexcitatory behavior after suffered from sevoflurane

Group	Label	NFκB concentration (ng/mL)	MDA concentration (nmol/mL)
Observation	Hyperexcitatory behavior	127.997	6.403
	Non hyperexcitatory behavior	131.215	5.887
Magnesium	Non hyperexcitatory behavior	125.234	5.416

Note: MDA: Malondialdehyde; NFκB: nuclear factor κB.

In vitro study

The view from the two-channel laser scanning confocal microscope was captured and showed the luminescence intensity for cytosol calcium levels from each wavelength. Figure 2 shows the examples of the pictures from each group. The animals in the observation group with post-anesthesia HEB had the highest mean calcium concentration (7147.022 nM) compared with those in the observation group without post-anesthesia HEB (5000.504 nM). The animals in the magnesium group without post-anesthesia HEB showed the lowest mean calcium concentration (3938.409 nM) (Table 2).

Figure 2.

Confocal microscope view on luminescence intensity of cytosol calcium levels in brain tissue of rats with hyperexcitatory behavior post sevoflurane exposure.

Note: The image shows the iluminescence at two different channels of a Confocal microscope which reflect cytosol calcium level according to the ratiometry property of the Fura-Red AM calcium that compares luminescence intensity of calcium-unbound and calcium bound. Left: the luminescence intensity in channel 1 (calcium-unbound). Right: the intensity of luminescence in channel 2 (calcium bound). HEB: Hyperexcitatory behavior.

Table 2.

Distribution of calcium content in brain tissue of rats with hyperexcitatory behavior after exposure to sevoflurane

Group	Label	Number of cells observed (n)	Median value (nM)	Interquartile range (nM)	Average value (nM)
Observation	Hyperexcitatory behavior	83	1949.165	122761.50	7147.022
	Non hyperexcitatory behavior	66	1739.537	42636.91	5000.504
Magnesium	Non hyperexcitatory behavior	72	1796.315	45435.96	3938.409

Note: Data were analyzed by the Kruskal-Wallis test followed by post hoc Mann-Whitney test. n: The number of cells being measured for luminescence Intensity (P = 0.003).

DISCUSSION

This study aimed to prove the relationship between increasing cytosol calcium levels and behavioral changes in experimental animal post-sevoflurane anesthesia exposure. Sevoflurane, a gamma amino butyric acid A receptor agonist, has an inhibitory effect throughout the entire anesthesia exposure, but at the same time, it stimulates InsP3 receptor, a ligand-gated Ca²⁺ release channel on intracellular Ca²⁺ signaling receptor, which releases calcium.9,10 The changes in the cytosol calcium level will induce a signal transmission in excitable and nonexcitable cells; thus promoting the excitatory pathway.8 The inhibitory activity will stay dominant throughout anesthesia; thus, the excitatory manifestation will not be seen. However, after the anesthesia agent exposure is ceased, the stimulation of the gamma amino butyric acid receptors will decrease, meanwhile a high cytosol level of calcium is sustained, causing an increase in the excitatory pathway. The rise in the calcium level does not directly cause a hyper excitatory response. Rather, it will decrease the threshold for stimulus response, causing an action potential, opening the voltage-gated calcium channel to release neurotransmitters in the synapses, manifesting as excitation-type delirium.

A study conducted by Lim et al.16 reported that HEB post-sevoflurane anesthesia occurred in immature rats aged 0–14 days. However, this phenomenon was not observed in mature rats, the study reported that the incidence of HEB subsides greatly after the age of 15 days. This finding was inconsistent with the findings of this study which show the occurrence of HEB on groups of young Sprague-Dawley rats, at the age of 2–5 weeks, as equal to 1–5 years old children. In this age group, the immature neurons have been replaced by the mature neurons. This study also provides evidence that post-anesthesia HEB is not related solely to the immaturity of the neurons, other factors must be taken into consideration.

In vitro experiments of the study started with decapitation of the heads of the experimental animals before harvesting the brains. The decapitation technique was chosen because it is an effective technique for euthanasia if performed by experienced animal laboratory personnel. Decapitation is a quick and humane method of euthanasia with less hypoxia as consciousness is likely to vanish within a few seconds after decapitation.23

The average normal results concerning the inflammation process and oxidative stress in this study tells us that the postanesthesia HEB was not correlated with either an inflammation reaction or oxidative stress due to exposure to volatile anesthesia, therefore considered as a physiological process. In a previous study, it was found that volatile anesthesia could induce neuroinflammation, marked by an increase of interleukin-6 in mouse brain tissue.24 Similarly, Yang et al.25 demonstrated the neuroinflammatory effect of sevoflurane in aged rats as shown by the increased levels of MDA, interleukin-17A, NFκB p65, and lower level of superoxide dismutase as an antioxidant. Some studies have demonstrated the neuroprotective effects of sevoflurane, where it was able to prevent oxidative stress events, thereby lowering the MDA level in the brain tissue.26,27

Intracellular calcium concentration in neurons is maintained around 100 nM, while it is 10,000-folds lower in the extracellular space and 1000-fold lower inside the intracellular storage of the ER. Volatile anesthetic agents were shown to cause excessive calcium release from ER into cytosol.27 The effects can be both neuroprotective and neurotoxic through different activation of inositol 1,4,5-triphosphate receptor (InsP3R) and calcium release from the storage of the ER. General anesthetics at low concentrations for short durations will induce endogenous neuroprotective mechanisms and provide neuroprotection via adequate activation of InsP3R, and moderate Ca²⁺ release from ER. On the other hand, general anesthetics at high concentrations for prolonged duration, like prolonged cerebral ischemia, become lethal stress factors, which induce neuronal damage by over activation of InsP3R, excessive and abnormal calcium release from ER.28

Intracellular calcium concentration has a very important role in triggering the release of neurotransmitters. In the physiologic process of neurons, the cytosol calcium level of 5-10 μΜ or 5000-10,000 nM contributes to a release in the neurons’ fast-acting neurotransmitters, causing excitation, such as glutamate and acetylcholine. Intracellular calcium is known to increase 100-fold during the action potential.29,30 An increase in the cytosol calcium concentration can also occur due to an influx of high concentration of extracellular calcium, causing the opening of the calcium channel, but not as significant as the release of calcium from the storage in the ER.31 The mean calcium concentration of animals in the Observation group with post-anesthesia HEB was 7147.022 nM, within the range of excitation trigger level for the release of neurotransmitter causing excitation. The Observation group with post-anesthesia HEB also had the highest cell membrane depolarization compared with the other groups.

Another aspect of the study relates to the group of animals receiving MgSO₄ intraperitoneally. The study showed that MgSO₄ decreased post-anesthesia HEB in the experimental animals; it also decreased the average cytosol calcium concentration. However, the calcium concentration was still observed above the resting level. High levels of intracellular calcium could damage the cell. However, there was no oxidative stress and neuron damage, characterized by the normal levels of MDA and NFκB. Aside from playing a role in the calcium reuptake process,32 MgSO₄ is known to block calcium’s exit to the extracellular cavity through voltage-sensitive calcium channel33 and inhibit the secretion of calcium from the ER as a non-competitive inhibitor against the InsP3-calcium channel.7

This study exhibited a persistently higher calcium level in the cytosol, even when the sevoflurane effect had subsided clinically during the emerging phase. The true elimination rate of sevoflurane cannot be determined by its pharmacological properties. However, rapid elimination of sevoflurane by 80% occurs through the lungs. A study by Farida et al.34 on cultured cells showed that the rise in the cytosol calcium level post-halothane anesthesia will subside within 15 minutes after cessation of anesthesia. Sevoflurane has a higher solubility in blood compared to halothane; this explains the shorter induction time and supposedly shorter elimination time. A delay in calcium reuptake into storage is thought to occur because of a delay in ER storage through sarcoplasmic-ER Ca²⁺-ATPase.35 Magnesium has an important role in intracellular calcium regulation; its administration is proven to lower the event of HEB post-volatile anesthesia.10 This is in accordance with a human study conducted by Lee et al.36

The limitation of this study was that we were unable to observe the changes in calcium cytosol concentration in a timelapse manner, because of facility limitations. However, the study provides another perspective on the pathophysiology of emergence delirium (Figure 3). If the mechanism of hyperexcitation post sevoflurane anesthesia is a physiological process, considering that there are no permanent changes in the cells, no inflammation and no oxidative stress response, the mechanism that occurred in the experimental animals could depict what really happens in humans during emergence delirium. The manifestation of HEB and the mechanism of cellular calcium regulation are comparable to what is seen in humans.37 Ultimately, this study can also explain how the administration of some drugs, such as intravenous anesthesia like propofol and benzodiazepine, α agonist agents and magnesium, can lower the risk of emergence delirium by prolonging the awakening process that will provide time for calcium to eliminate back to the storage in reticulum endoplasmic, or to increase the elimination of calcium from the cytosol. It is concluded from this study that the level of intracellular calcium in rats experiencing hyperexcitatory behavior after exposure to sevoflurane was higher and might have a role in the mechanism. Magnesium may attenuate the occurrence of this phenomenon. The findings of this study will open further studies for possible prophylactic approaches to emergence delirium.

Figure 3.

Pathophysiology pathway of sevoflurane exposure and emergence agitation.

Note: Ca²⁺: Calcium ion; InsP3: inositol 1,4,5-triphosphate; SERCA: sarco/ endoplasmic reticulum Ca²⁺-ATPase.

Additional file

Additional Table 1: The wavelength of two-channel laser scanning confocal microscope.

Funding Statement

Funding: This study was supported by the Department of Anesthesiology and Intensive Care, Faculty of Medicine, Indonesia University/ Cipto Mangunkusumo Hospital.