Effects of sugammadex and rocuronium mast cell number and degranulation in rat liver
We investigated the effect of rocuronium- and sugammadex-induced mast cell increase and degranulation in rat portal triads. Forty-two rats, in six groups, received either rocuronium 1 mg.kg−1; sugammadex 15 mg.kg−1; sugammadex 100 mg.kg−1; rocuronium 1 mg.kg−1 and 5 min later, sugammadex 15 mg.kg−1; rocuronium 1 mg.kg−1 and 5 min later, sugammadex 100 mg.kg−1; or isotonic saline. Total mast cell numbers were significantly higher with rocuronium only, than in all other groups (p < 0.003), although in all active groups, the number was greater than the control. Total mast cell number was significantly higher with rocuronium and low-dose sugammadex compared with low-dose sugammadex only. The number of tryptase-positive mast cells with rocuronium only was significantly higher than in all other groups (p < 0.003). Tryptase-positive mast cell numbers in both groups receiving both rocuronium and sugammadex were significantly higher compared with both groups receiving sugammadex only. Rocuronium increased mast cell numbers, and degranulation was mitigated by sugammadex. These results suggest that sugammadex may be beneficial in treatment of rocuronium-induced anaphylaxis.
Anaphylactoid and anaphylactic reactions occur during general anaesthesia [1, 2]. Non-depolarising neuromuscular blocking agents are the most frequent cause of these (58–69%), and rocuronium is frequently implicated (in 43% of cases) [3–6].
Sugammadex is a recently developed γ-cyclodextrine, designed to bind non-depolarising neuromuscular blocking agents like rocuronium . By encapsulating rocuronium molecules in plasma and detaching them from the neuromuscular junction, sugammadex rapidly reverses rocuronium-induced neuromuscular blockade and eliminates circulating rocuronium molecules via the renal route, thereby preventing their metabolisation [8, 9]. Recent studies report successful use of sugammadex in the treatment of anaphylactic reactions induced by rocuronium, although the mechanism of action is not clear [10, 11]. However, it has been reported that sugammadex itself can induce an anaphylactic reaction .
Mast cells are immune cells residing in connective tissue containing large metachromatic granules [13, 14]. Tryptase is a neutral protease considered as a marker of mast cell degranulation . Tryptase, being the most abundant protein stored in human mast cell granules, probably has a role in regulating allergic and inflammatory reactions. To date, no study investigating the effects of rocuronium and sugammadex on mast cells has been published. We used a rat model to investigate rocuronium-induced increases in mast cell number and degranulation in the liver portal triad and the effects of sugammadex on this model, using light microscopy and immunohistochemisrty.
All experiments were conducted according to our National Institute of Health Guide for the Care and Use of Laboratory Animals. The study protocol was approved by the Local Ethics Committee for Animal Experiments of Rize University, Turkey. Forty-two 150–180-day-old Sprague-Dawley male rats weighing 300–350 g were fed with standard pellet diet and water ad libitum (Veterinary Laboratory, Erzurum, Turkey). Room temperature (22–25 °C) and humidity (50–55%) were monitored daily. Lighting was provided using cool white fluorescent lamps, with 12-h light and dark cycles (06.00–18.00). The animals were divided into six groups (n = 7), receiving one of: rocuronium 1 mg.kg−1 (Esmeron; Organon, Istanbul, Turkey; Group R); sugammadex 15 mg.kg−1 (Bridion; Schering-Plough Corporation, Oss, Netherlands; Group S15); sugammadex 100 mg.kg−1 (Group S100); rocuronium 1 mg.kg−1 and 5 min later, sugammadex 15 mg.kg−1 (Group RS15); rocuronium 1 mg.kg−1 and 5 min later, sugammadex 100 mg.kg−1 (Group RS100); or isotonic saline as control (Group C). All drugs were administered intravenously in 1-ml volumes via the tail vein.
Rats in Group R received a tracheotomy under ether anaesthesia and their lungs were ventilated until resolution of spontaneous ventilation. After resolution of spontaneous ventilation, the tube was removed and the tracheotomy site sutured. The lungs of rats in Groups RS15 and RS100 were ventilated via mask until resolution of spontaneous ventilation following administration of sugammadex. All rats were observed for 24 h following the experiment and then underwent euthanasia with ether. After ether euthanasia, the animals received intracardiac perfusion with 4% formaldehyde solution at room temperature. Whole liver tissue was extracted and stored in 10% formalin overnight at 4 °C. Specimens were washed in water for 12 h on the following day and treated in graded alcohol and xylene and embedded into liquid paraffin. Suitable tissue sections were cut at a thickness of 4–6 μm using a microtome (Leica RM2255; Leica Microsystems, Wetzlar, Germany) and stained with haematoxylin–eosin and toluidine blue. These were examined under a light microscope (Olympus BX51, Olympus Corporation, Tokyo, Japan) with an attached digital camera (Olympus DP72, Olympus Corporation, Tokyo, Japan) at 20× and 40× magnifications. Tryptase-positive cells were investigated using streptavidin-biotin-peroxidase staining (Monoclonal Mouse Anti-Human Mast Cell Tryptase, Clone AA1, M 7052; Dako, Carpinteria, CA, USA). Two slides from each rat were prepared and five portal triads were examined from each slide. Two histologists blinded to the group graded immunoreactivity. Total and tryptase-positive (activated) mast cells present in all groups were counted.
Blood samples were withdrawn from the cardiac chambers to measure serum aspartate and alanine aminotransferase (AST and ALT) concentrations. Blood AST and ALT concentrations were determined by a photometric method using the Architect C 16000 chemistry analyser (Abbott Laboratories, Santa Clara, CA, USA).
Results were analysed using SPSS software (SPSS 12 for Windows; IBM, Chicago, USA). Data were tested for normal distribution using the Kolmogorov–Smirnov test. Results were analysed using the Kruskal–Wallis test. Groups were separately compared using the Mann–Whitney U-test (with post hoc Bonferroni’s correction). The p value was adjusted for multiple comparisons by dividing 0.05 by 15, and p values < 0.003 were considered statistically significant.
None of the rats showed signs of infection, anaphylactic shock or a change in feeding pattern during the study period.
Total mast cell numbers in Group R (rocuronium only) were significantly higher than in all other groups (p < 0.003). Total mast cell numbers in Groups S15, S100, RS15 and RS100 were also significantly higher than in the control group. Total mast cell number was significantly higher in Group RS15 (rocuronium and low-dose sugammadex) compared with Group S15 (low-dose sugammadex only; Table 1).
Table 1. Total and tryptase-positive mast cell numbers and ALT and AST concentrations in rats receiving rocuronium 1 mg.kg−1 (Group R), sugammadex 15 mg.kg−1 (Group S15), sugammadex 100 mg.kg−1 (Group S100), rocuronium 1 mg.kg−1 and 5 min later sugammadex 15 mg.kg−1 (Group RS15), rocuronium 1 mg.kg−1 and 5 min later sugammadex 100 mg.kg−1 (Group RS100) or isotonic saline as control (Group C). Values are mean (SD).
|Mast cells; n||256 (13)*||121 (6)||130 (15)||153 (19)||133 (11)||93 (11)†|
|Tryptase-positive mast cells; n||89 (11)*||29 (9)||24 (5)||57 (8)||50 (8)||17 (8)†|
|ALT; U.l−1‡||27.4 (7.4)||28.6 (7.1)||28.1 (7.5)||30.4 (12.7)||31.7 (14.4)||22.6 (5.1)|
|AST; U.l−1§||81.1 (23.2)||77.0 (19.8)||71.4 (22.9)||77.7 (26.9)||87.6 (24.8)||60.3 (11.9)|
The number of tryptase-positive mast cells in Group R were significantly higher than in all other groups (p < 0.003). Tryptase-positive mast cell numbers in Groups RS15 and RS100 were significantly higher compared with Groups S15 and S100. Comparison of tryptase-positive mast cell numbers between Groups S15 and S100, and between Groups RS15 and RS100 showed no significant difference. Tryptase-positive mast cell numbers in Groups RS15 and RS100 were significantly higher compared with the control group (Table 1). Levels of AST and ALT in all groups were within reference ranges (Table 1).
All rats in our study groups showed increased mast cell numbers compared with the control group; this was greatest in rats receiving rocuronium only, suggesting that both rocuronium and sugammadex increased mast cell numbers in hepatic connective tissue. Rats receiving the same dose of rocuronium and additional sugammadex (Groups RS15 and RS100) also showed increased mast cell numbers; however, the degree of increase was lower compared with rats receiving rocuronium only. There is some suggestion here that sugammadex may dose-dependently reduce rocuronium’s effect on mast cell numbers, which merits further investigation.
Menéndez-Ozcoidi and colleagues reported an allergic reaction following administration of sugammadex 3.2 mg.kg−1 to reverse neuromuscular blockade induced with rocuronium . In our study, rats receiving sugammadex alone showed increased mast cell numbers compared with the control group. This suggests that sugammadex causes an increase in mast cell numbers and may predispose to allergic reactions. However, the sugammadex-induced increase was significantly lower than the rocuronium-induced increase.
Tryptase-positive mast cell numbers were significantly higher in rats receiving rocuronium alone compared with all other groups. Rats receiving the same dose of rocuronium and additional sugammadex (Groups RS15 and RS100) also showed increased mast cell degranulation; however, the degree of degranulation was less prominent compared with rats receiving rocuronium only. In Group RS100, which received rocuronium and a high dose of sugammadex, mast cell degranulation was less marked than in Group RS15, which received rocuronium and a lower dose of sugammadex. This suggests that sugammadex may dose-dependently inhibit rocuronium-induced mast cell degranulation, again something needing further investigation, as our numbers and range of doses are insufficient to be conclusive.
In this study, tissue preparation and mast cell counts were performed 24 h after drug administration. This was decided upon after consideration was given to the pharmacokinetics of rocuronium and sugammadex. After bolus administration, rocuronium is eliminated primarily by the liver. Plasma concentration of rocuronium rapidly falls due to hepatic uptake and excretion in the bile. About a third of the remaining drug in circulation is excreted in the urine in 24 h [16, 17]. Consequently, rocuronium remains in the body for at least 24 h. If sugammadex is administered after a bolus dose of rocuronium, sugammadex molecules rapidly encapsulate the circulating rocuronium molecules. As the plasma concentration of rocuronium falls, rocuronium molecules absorbed by the liver and bound to neuromuscular junction return to the plasma and are continuously encapsulated by circulating free sugammadex molecules. Over a 24-h period, 96–97% of the rocuronium–sugammadex complex is excreted in the urine [9, 10].
Clarke and colleagues used a cutaneous model of anaphylaxis to investigate the efficacy of sugammadex in preventing or mitigating rocuronium-induced allergic reactions in patients known to be sensitive to rocuronium . They showed that rocuronium-induced reactions were attenuated, but not completely abolished, by sugammadex administered 2 min later. Similarly, our study shows that administration of sugammadex 5 min later attenuated the rocuronium-induced increase in tryptase concentration in rat liver. As Clarke et al. note, the tryptase concentration in the tissue and plasma increases at the beginning of an anaphylactic reaction. Serum or tissue tryptase levels can be measured by ELISA or total or specific IgE [19, 20]. Lack of this measurement is the main limitation of our study. If tissue or plasma concentrations of specific IgE could be measured serially, the demonstration of an increase or decrease in concentration would be helpful in further clarifying sugammadex’s effect on the allergic response.
Harper emphasised in his commentary  that rocuronium must be separated from IgE-binding sites and eliminated rapidly so that its plasma concentration will quickly fall, preventing further interaction with IgE receptors via encapsulation. Leysen and colleagues, sharing the same concerns, designed an in vitro study  that showed that rocuronium encapsulated in sugammadex is unable to activate basophils. Both Leysen et al.’s and our current findings suggest that administration of sugammadex attenuates the activation of mast cells.
No external funding or competing interests declared.