Effect of Chronic Exposure to Dexamethasone on Rocuronium-induced Neuromuscular Blockade and Sugammadex Reversal: an in Vivo Study on Rats

Background: Chronic exposure to glucocorticoids is associated with resistance to nondepolarising neuromuscular blocking agents. Therefore, we hypothesised that sugammadex-induced recovery in subjects with chronic exposure to dexamethasone was faster than that in subjects without dexamethasone exposure. Objective: To evaluate the recovery prole of rocuronium-induced neuromuscular blockade after sugammadex administration in rats. Design: An in vivo study on rats. Animals: Thirty-six male Sprague-Dawley rats. Intervention: Sprague–Dawley rats were allocated to three groups (dexamethasone group, control group, and pair-fed group) for the in vivo study. Dexamethasone group received daily intraperitoneal injections of dexamethasone 500 μg kg -1 or 0.9% saline for 15 days. On the sixteenth day, 3.5 mg kg -1 of rocuronium was administered to achieve complete neuromuscular blockade. Main outcome measures: The recovery time to a train-of-four ratio Results: There were no signicant differences in the recovery time to train-of-four ratio to 0.9 among the groups (P = 0.531). The time to second twitch of train-of-four recovery that indicated the duration of rocuronium-induced neuromuscular blockade was signicantly shorter in Group D than in Groups C and P (P = 0.001). Conclusion: As previously reported, resistance to rocuronium was observed in rats with chronic exposure to dexamethasone. However, the neuromuscular recovery time after sugammadex administration was not signicantly different between groups.


I. Introduction
Glucocorticoids have been prescribed for diseases caused by in ammation, such as chronic obstructive pulmonary disease, allergies, rheumatoid arthritis, osteoarthritis, in ammatory bowel diseases, eczema, and other allergic skin conditions. It is also used to achieve immunosuppression after organ transplantation. Glucocorticoids are used in all medical specialties. However, they are a double-edged sword, as anticipated clinical effects could be accompanied with adverse outcomes despite proper dosage and duration of use. Neuromuscular blocking agents (NMBAs) are important for maintaining good surgical conditions and mechanical ventilation under general anaesthesia. Long-term glucocorticoid-induced resistance to numerous nondepolarising NMBAs, such as atracurium, vecuronium, and rocuronium, has been observed in studies [1,2].
Chen et al. reported that chronic dexamethasone treatment increased the percentage of immature subunits (γ-subunit) of the nicotinic acetylcholine receptor (nAChR) and increased the expression of nAChRs in Sprague-Dawley rats treated with daily dexamethasone for 14 days [1]. These phenomena would increase the amount of NMBAs required to competitively block nAChRs. In addition, chronic exposure to glucocorticoids is known to lead to a reduction in type II bres in the muscle, which are more sensitive to NMBAs than type I bres [2]. These factors could lead to resistance to NMBAs.
Sugammadex is a cyclodextrin molecule with an ability to encapsulate lipophilic compounds [3]. It selectively binds to the rocuronium molecule, which has a steroidal nucleus. This binding prevents rocuronium from binding to nAChRs and allows rapid reversal of neuromuscular blockade (NMB). Considering the resistance to NMBA caused by chronic exposure to dexamethasone, it is reasonable to assume that sugammadex-induced recovery of NMB may be affected.
However, clinical studies investigating dexamethasone long term treatment with dose known to cause muscle atrophy are still unavailable and large-scale investigations are not feasible due to ethical constraints. Therefore, we designed speci c environmental conditions of dexamethasone long term treatment under in vivo conditions to demonstrate the effects of rocuronium and sugammadex using the animals We hypothesised that sugammadex-induced NMB recovery in rats with chronic dexamethasone exposure is faster than that in rats without dexamethasone exposure. The objective of this study was to evaluate the recovery pro le of rocuronium-induced NMB after chronic exposure to dexamethasone using sugammadex as a reversal agent, since the in vivo effect of sugammadex reversal on rocuroniuminduced NMB in subjects with chronic dexamethasone exposure has not been investigated yet.
The primary outcome was the recovery time to a train-of-four (TOF) ratio ≥ 0.9 (TTOFr), which is the time it takes for the TOF ratio (TOFr) to recover to 0.9 or higher after the injection of sugammadex. Secondary outcomes were time of T1 (the rst twitch of TOF) recovery to 95% (TT1) and the recovery index (RI). TT1 is the time taken for the rst twitch of TOF to recover to 95% of the baseline T1 after the injection of sugammadex. RI is the time taken for T1 of TOF to recover from 25-75% of the baseline T1. There are numerous controversies regarding the calculation of the number of samples in animal experiments. We did not estimate the sample size statistically. Previous studies have conducted experiments with about 10 animals per group to achieve statistically signi cant results [5][6][7]. In a previous study that investigated the effects of dexamethasone on sugammadex reversal in rocuroniuminduced NMB in animals, it was suggested that 10 animals in one group are su cient to produce signi cant results [6,8,9]. To allow for attrition, thirty-six adult male Sprague-Dawley rats (7-week-old; weighing 213-253 g) were randomly divided into three groups (n = 12 per group). Sorting was accomplished using a random number generator in Microsoft Excel 2013 (Microsoft, Redmond, WA, USA). To apply the chronic dexamethasone exposure with a dose previously shown to cause muscle atrophy, the dexamethasone group (Group D) received daily intraperitoneal injections of 500 µg kg −1 dexamethasone disodium phosphate (Yuhan, Seoul, Republic of Korea) for 15 days [1,2,10]. One millilitre of 0.9% saline was used to suspend 500 µg of dexamethasone. Thus, rats weighing 213-253 g were injected with 106.5-126.5 µg of dexamethasone in a 0.2-0.25 mL volume. The control group (Group C) only received an equivalent volume of 0.9% saline daily for 15 days. The rats in the pair-fed group (Group P) were fed daily the same amount of food as was consumed by rats in Group D for 15 days. All treatments were done in the laboratory.
The authors weighed the amount of food consumed by Group D daily and provided the same amount of food to Group P daily. Group P was pair-fed with the Group D for 15 days to evaluate whether muscle dysfunction following dexamethasone treatment was caused by anorexia typically associated with glucocorticoid therapy. Food was available ad libitum to rats in Groups C and D. Weights of the rats were recorded daily, and dexamethasone doses were adjusted in accordance with change in body weight. Water was available ad libitum for all groups. All mice were bred in the laboratory animal breeding room at the Laboratory Animal Research Center, Asan Institute for Lifesciences. The animals were housed in an individually ventilated cage system (Tecniplast, USA) under speci c pathogen-free conditions. The rats were raised at a constant temperature of 22°C, humidity 50 ± 10%, laboratory rodent chow and were maintained under a regular diurnal (12-h light and 12-h dark) cycle. All injections were administered at the same time of the day. Figure 1 summarises the treatments.

General surgical procedures
Twenty-four hours after the nal drug administration, the rats were anaesthetised with an intraperitoneal injection of Alfaxan TM (Jurox Pty. Limited, New South Wales, Australia) at 40 mg kg −1 of body mass, and an adequate depth of anaesthesia was con rmed by the absence of a withdrawal response to toe clamping [11]. When there was a withdrawal response, an additional 10-20 mg kg −1 was injected if necessary. The animals were tracheotomised, mechanical ventilation was applied to maintain normal breathing throughout the surgery, and the jugular vein was catheterised to inject drugs. Body temperature was monitored using an oesophageal temperature probe (Regulation to 37 ± 1°C), and a warming pad and light source were used to maintain proper body temperature. The tibialis anterior muscle was exposed, and the distal part of the tendon was tied with 3-0 black silk. It was then connected to a force displacement transducer (Grass FT03, Grass Instrument Co., Quincy, Massachusetts, USA) to measure the isometric contraction of the tibialis anterior muscle at a resting tension of 2 g. The sciatic nerve was exposed and connected to the bipolar platinum electrodes to evaluate neuromuscular transmission.

Assessment of neuromuscular transmission
Using a nerve stimulator (S88, Grass) and stimulation isolation unit (SIU5, Grass), TOF stimulation (frequency 2 Hz, duration 0.2 ms) consisting of four supramaximal square-wave pulses were applied to the sciatic nerve via the bipolar platinum electrodes every 12 s throughout the study. Muscle contraction responses were recorded and digitalised with a PowerLab acquisition system (ADInstruments, Austin, Texas, USA) and stored in LabChart7 (ADInstruments, Colorado Springs, CO, USA), which is a computer with data charting software. In all groups, contraction responses were stabilised for at least 10 min after the initiation of TOF stimulation. The height of T1 was measured as the baseline T1 after 10 min of stabilisation. After administration of 3.5 mg kg −1 of rocuronium (Esmeron TM , MSD, Oss, The Netherlands), a dose ensuring that complete NMB is achieved in rats [1], via the jugular vein catheter, complete NMB was induced [12]. The time from rocuronium injection to the appearance of the second twitch of TOF (TT2) was recorded. When T2 appeared, sugammadex (Bridion TM , MSD, Oss, The Netherlands) 0.5 mg kg −1 was administered, and the TTOFr, which was our primary outcome, was recorded. RI and TT1 were recorded as secondary outcomes. Figure 2 summarises the overall experiment.

Specimen measurement
On completion of the in vivo study, the rats were sacri ced for specimen examination.

Data and statistical analysis
The primary outcome of this study was the TTOFr. The secondary outcomes were the TT1 and RI. Data are expressed as the mean ± standard deviation or median [Interquartile range] unless otherwise speci ed. Quantile-quantile plots were used to assess the normality assumption. One-way analysis of variance followed by the Tukey post hoc test was applied to analyse the weight of rats, temperature, weight of the tibialis anterior muscle, TT2, TTOFr, TT1, and RI. The Kruskal-Wallis test was used to analyse the length and width of the tibialis anterior muscle. Statistical signi cance was set at P values < 0.05, and all statistical tests were two-sided. SAS statistical software (version 9.3; SAS Institute Inc., Cary, North Carolina, USA) was used for statistical analysis.
Iii. Results

Animal and specimen data
Thirty-six rats were allocated to three groups, with 12 rats assigned to each group. One rat in Group C was administered with rocuronium overdose and another rat died during the surgical procedure; thus, data from the two rats could not be used. We could not use data of two rats in Group D because of a ventilator breakdown resulting in respiratory failure in the rat, and a computer system shutdown during the recovery process resulting in recording error. In Group P, data of two rats could not be used. Incomplete NMB even after administration of the proper dosage of rocuronium with su cient time for onset of the drug, and another rat showed incomplete recovery of TOFr. Therefore, a total of 30 rats (10 each in Groups C, D, and P) were included in the analysis (Figure 3).
There were no signi cant differences in the initial body weight before the experimental treatment, but the nal body weight after the experimental treatment signi cantly differed between the groups (Table 1). Food intake decreased markedly among the rats in Group D, as previously reported [13,14]. The size and weight of the tibialis anterior muscles were smaller in Group D than in Groups C and P. Though rats in Groups D and P were fed the same amount of food, the degree of weight loss, muscle size reduction, and muscle weight loss were greater in Group P. Data are presented as mean ± standard deviation or median [Interquartile range]; Group C, control group; Group D, dexamethasone group; Group P, pair-fed group. Group C received only the amount of 0.9% saline received by Group D each day. Group D received a daily intraperitoneal injection of 500 µg kg −1 dexamethasone, suspended in 1 mL of 0.9% saline. Group P was fed daily with the same amount of food consumed by group D. Body temperature was monitored with an oesophageal temperature probe and maintained with a warming pad and a light source for warming within the range of normal body temperature. * P < 0.001 vs. Group C. † P < 0.001 vs. Group P. One-way analysis of variance was performed followed by the Tukey post hoc test.

NMB induction
After administration of 3.5 mg kg −1 (estimated 2-fold ED 90 ) [15] of rocuronium via the jugular vein, complete NMB was induced in each group.

Recovery pro les
TTOFr, the primary outcome, was not signi cantly different between the groups (4.3 ± 2.3, 4.0 ± 3.0, and 3.1 ± 1.8 min, P = 0.531 in Groups C, P, and D, respectively) ( Table 2). TT1 was signi cantly shortened in Group D than in Groups C and P (3.6 ± 2.1 min vs. 4.2 ± 2.2 and 4.4 ± 2.5 min, P = 0.001, respectively) ( Table 2). However, RI was not signi cantly different between the groups (1.5 ± 0.6, 2.2 ± 1.8, and 1.3 ± 0.6 min, in Groups C, P, and D, P = 0.531, respectively) ( Table 2). TT2, which shows the duration of rocuronium-induced NMB, was signi cantly shortened in group D when compared with that in Groups C and P (2.9 ± 1.0 min vs. 5.0 ± 1.1 and 5.1 ± 1.4 min, P = 0.001, respectively), while no signi cant difference was observed between Groups C and P (P = 0.996).  Data are presented as mean ± standard deviation; Group C, control group; Group D, dexamethasone group; Group P, pair-fed group. Group C received only the amount of 0.9% saline received by Group D each day. Group D received a daily intraperitoneal injection of 500 µg kg −1 dexamethasone, suspended in 1 mL of 0.9% saline. Group P was fed daily with the same amount of food that was consumed by Group D.
Times and recovery indices are presented as minutes; TOF, train-of-four; T1, the rst twitch of train-of-four; T2, the second twitch of train-of-four; TT2, the time from rocuronium injection to appearance of T2; TTOFr, time taken for the TOF ratio to recover to 0.9 or higher after injection of sugammadex; TT1, the time taken for the T1 to recover to 95% of the baseline T1 after injection of sugammadex; RI, recovery index (the time taken from T1 of 25% of the baseline T1 to T1 of 75% of the baseline T1). * P = 0.001 vs.
Group C. † P = 0.001 vs. Group P. One-way analysis of variance was performed followed by the Tukey post hoc test.

Vi. Discussion
The primary outcome of this study was that chronic exposure to dexamethasone, while inducing resistance to rocuronium, did not have any signi cant effect on sugammadex reversal in this in vivo study on rats.
This result was contrary to our hypothesis. We considered the following reasons for this unexpected result. First, in rats, the circulation time is faster than that in humans. As the speed of the reversal was extremely fast, it was hard to determine differences in the recovery pro les that may have existed. The use of a single twitch stimulation would have been better. As TOF stimulation usually occurs every 10-15s, 1 Hz single twitch stimulation could have provided the more appropriate resolution to identify the fast recovery from deep neuromuscular block [15].
Second, we used only healthy young rats in this study. Patients in the intensive care unit may develop resistance to NMBAs during chronic treatment with glucocorticoid; additionally, co-administration of corticosteroid and NMBA can lead to prolonged weakness and even acute myelopathy [16]. Hence, the use of sugammadex as an NMB reversal agent might have resulted in different recovery pro les if rats sick enough to receive ICU care were used in the study.
Third, chronic exposure to dexamethasone is known to induce nAChR upregulation and expression of the immature form of the receptor subunit, causing resistance to NMBA. However, sugammadex encapsulates NMBA molecules, regardless of the receptor's sensitivity. This may be one of the reasons for no signi cant difference in the recovery pro le with administration of sugammadex, despite the shorter duration of NMB in the group with long-term dexamethasone exposure. Use of anticholinesterases such as pyridostigmine and neostigmine as reversal agents would have resulted in different recovery pro les.
There are few in vivo studies on rats that demonstrate the effect of chronic dexamethasone exposure on sugammadex reversal. Previous studies have reported that resistance to rocuronium-induced NMB was observed in rats with chronic exposure to dexamethasone. The effects of glucocorticoids can be categorised as short-term and long-term treatment effects. The presynaptic effects have been observed during short-term treatment. Synthesis and increased release of acetylcholine have been observed [7].
The nAChR in the muscle forms a heteropentamer consisting of two alpha, one beta, and one delta subunit with one gamma subunit in the fetal AChR isoform, which is replaced by an epsilon subunit in the adult AChR isoform. During long-term treatment, changes occur in the nAChRs subunit [1]. The epsilon subunit turns into the gamma subunit, which is an immature form that is resistant to NMBAs. Functional upregulation of the nAChRs is observed during both short-term and long-term glucocorticoid treatment, and this has been documented in burns and immobilisation injury [1,17,18].
According to a study by Lee et al., resistance to nondepolarising NMBAs, which occurs after immobilisation, might be related to the upregulation of α7-nAChRs [17]. It has also been shown that α7-nAChRs expression occurs after protracted dexamethasone treatment [19].
One of the limitations is the dosage of sugammadex used in the study. There is no consensus on the recommended dose of sugammadex for an in vivo study using rats, as there are few such studies [15,20]. Therefore, the dose of sugammadex was determined in a pilot study. The dose of sugammadex that resulted in faster recovery without resulting in an extremely rapid recovery from NMB was chosen. However, when a reduced dose of sugammadex was used, there was no signi cant difference in TTOFr.
However, the concentration (dexamethasone 500 µg kg −1 ) used in this study far exceeds the typical clinical doses. In clinical concentrations of steroids, there would be no effect on sugammadex reversal.
In conclusion, chronic exposure to dexamethasone while inducing resistance to rocuronium did not have any signi cant effect on sugammadex reversal in this in vivo study on rats.

Declarations
Availability of data and materials The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Figure 1
Flow diagram of the treatment IPI, intraperitoneal injection; Group C, control group; Group D, dexamethasone group; Group P, pair-fed group. Group C received the same volume of 0.9% saline received by Group D each day. Group D received a daily IPI of 500 μg kg-1 dexamethasone suspended in 1 mL of 0.9% saline. Food and water were provided ad libitum. Group P received the same volume of 0.9% saline received by Group D each day and fed daily with the same amount of food and water as consumed by Group D.

Figure 2
Flow diagram of the experiment Groups included: control group (Group C), dexamethasone group (Group D), and pair-fed group (Group P); NMB, neuromuscular blockade; T2, the second twitch of train-of-four stimulation; IVI, intravenous injection; IPI, intraperitoneal injection. Group D received a daily IPI of 500 μg kg-1 dexamethasone suspended in 1 mL of 0.9% saline. Food and water were provided ad libitum. Group C received the same volume of 0.9% saline received by Group D each day. Food and water were provided ad libitum. Group P received the same volume of 0.9% saline received by Group D each day and were fed daily with the same amount of food and water as consumed by Group D.

Figure 3
Flow chart of experimental procedure Group C, control group; Group D, dexamethasone group; Group P, pair-fed group; S-D, Sprague-Dawley; NMB, neuromuscular blockade, TOFr; train-of-four ratio.