Etomidate-Induced myoclonus in Sprague‒Dawley rats involves the activation of neocortical Calpain-2 and its decrement on KCC2 protein

Background: Etomidate-induced myoclonus has become a pressing clinical problem with an incidence of 50-80%. The underlying mechanism involves neocortical glutamate accumulation and N-methyl-d-aspartate (NMDA) receptor activity. However, the therapeutic target remains uncertain.

Methods: Adult male Sprague-Dawley (SD) rats were injected with etomidate (1.5 mg/kg), propofol (11.8 mg/kg), and lidocaine (4.0 mg/kg) plus etomidate (1.5 mg/kg), etomidate (3.8 mg/kg), etomidate (6.0 mg/kg) through the tail vein and behavioral scores of the rats were recorded within 5 min after anesthesia to establish the model of etomidate-induced myoclonus and to observe the dose dependence. The in vitro Western blot analysis of NKCC1 and KCC2 proteins and the regulatory effect of N-methyl-d-aspartate (NMDA) receptor were performed to find the potential target of etomidate-induced myoclonus or excitability. Additionally, to verify whether calpain-2 is involved in the process of regulatory effect of NMDAR on the cleavage of KCC2 protein during etomidate-induced myoclonus, muscular tension and KCC2 protein were analyzed in rats microinjected with calpain-2 inhibitor (MDL-28170) or MDL-28170 + NMDA in the neocortical motor cortex during etomidate anesthesia. Finally, MDL-28170 or vitamin E was injected intravenously before etomidate, the muscular tension, KCC2 protein and duration of loss of righting reflex (LORR) of rats were evaluated to verify the neuroprotective effect of vitamin E.

Results: Etomidate significantly increased the mean behavioral score at different time points compared with the propofol and lidocaine + etomidate groups within 5 min after anesthesia; the mean behavioral score decreased at different time points with increasing dose of etomidate. 0.5 µM ( 0.73 ± 0.18 vs. 1.04 ± 0.17, n = 6, p = 0.0096) and 1 µM (0.73 ± 0.24 vs. 1.03 ± 0.14, n = 6, p = 0.0077) etomidate induced the decrement of neocortical KCC2 protein compared to the control group. NMDA activated but 2-amino-5-phosphonopentanoic acid (AP5) inhibited 0.5 and 1 µM etomidate-induced decrement of neocortical KCC2 protein. MDL-28170 microinjected into the neocortex during etomidate anesthesia not only inhibited the decrement of KCC2 protein but also blocked the muscular tension induced by etomidate alone or etomidate plus NMDA. Intravenous injection of vitamin E prevented etomidate-induced muscular tension and decrement of the KCC2 protein.

Conclusion: Calpain-2 was involved in the process of etomidate-induced myoclonus and NMDAR activity by promoting the decrement of KCC2 protein and exerting the excitability. Vitamin E, as a natural antioxidant, can effectively prevent etomidate-induced myoclonus and does not affect recovery after etomidate anesthesia in rats.

Calpain-2; Etomidate; KCC2; Myoclonus; Vitamin E.