Dexamethasone as adjuvant to bupivacaine prolongs the duration of thermal antinociception and prevents bupivacaine-induced rebound hyperalgesia via regional mechanism in a mouse sciatic nerve block model

Abstract

Background: Dexamethasone has been studied as an effective adjuvant to prolong the analgesia duration of local anesthetics in peripheral nerve block. However, the route of action for dexamethasone and its potential neurotoxicity are still unclear.

Methods: A mouse sciatic nerve block model was used. The sciatic nerve was injected with 60ul of combinations of various medications, including dexamethasone and/or bupivacaine. Neurobehavioral changes were observed for 2 days prior to injection, and then continuously for up to 7 days after injection. In addition, the sciatic nerves were harvested at either 2 days or 7 days after injection. Toluidine blue dyeing and immunohistochemistry test were performed to study the short-term and long-term histopathological changes of the sciatic nerves. There were six study groups: normal saline control, bupivacaine (10mg/kg) only, dexamethasone (0.5mg/kg) only, bupivacaine (10mg/kg) combined with low-dose (0.14mg/kg) dexamethasone, bupivacaine (10mg/kg) combined with high-dose (0.5mg/kg) dexamethasone, and bupivacaine (10mg/kg) combined with intramuscular dexamethasone (0.5mg/kg).

Results: High-dose perineural dexamethasone, but not systemic dexamethasone, combined with bupivacaine prolonged the duration of both sensory and motor block of mouse sciatic nerve. There was no significant difference on the onset time of the sciatic nerve block. There was "rebound hyperalgesia" to thermal stimulus after the resolution of plain bupivacaine sciatic nerve block. Interestingly, both low and high dose perineural dexamethasone prevented bupivacaine-induced hyperalgesia. There was an early phase of axon degeneration and Schwann cell response as represented by S-100 expression as well as the percentage of demyelinated axon and nucleus in the plain bupivacaine group compared with the bupivacaine plus dexamethasone groups on post-injection day 2, which resolved on post-injection day 7. Furthermore, we demonstrated that perineural dexamethasone, but not systemic dexamethasone, could prevent axon degeneration and demyelination. There was no significant caspase-dependent apoptosis process in the mouse sciatic nerve among all study groups during our study period.

Conclusions: Perineural, not systemic, dexamethasone added to a clinical concentration of bupivacaine may not only prolong the duration of sensory and motor blockade of sciatic nerve, but also prevent the bupivacaine-induced reversible neurotoxicity and short-term "rebound hyperalgesia" after the resolution of nerve block.

Fig 1. Paw withdrawal latency behavioral changes in response to thermal stimulation after sciatic nerve block with various medications.

* indicates P<0.05 when compared with other groups. # indicates P<0.05 when compared with baseline control. The data were reported as mean ± SEM. NS, normal saline; Bup, bupivacaine; Dexa, dexamethasone; i.m., intramuscularly.

Fig 2. Motor recovery of mouse sciatic nerve block with various medication.

* indicates P<0.05 when compared with other groups. # indicates P<0.05 when compared with baseline control. The data were reported as mean ± SEM. NS, normal saline; Bup, bupivacaine; Dexa, dexamethasone; i.m., intramuscularly.

Fig 3. Microscopic changes of sciatic nerves after sciatic nerve block.

3A. istomorphological changes of paraffin-embedded sciatic nerves on day 2 and day 7 after injection (400×, toluidine blue staining). Nerves appeared normal on day 2 and day 7 after injection in most groups, while nerves in the bupivacaine treated group (red box) showed fiber degeneration with vague myelin sheath appearance (arrows) at 2 days after injection; 3B. Percentage of demyelinated axons in sections (n = 5 per group). The percentage of unmyelinated axons in bupivacaine group was significant higher on post-injection day 2 than on day 7 (*P<0.05), while no significant difference was observed in other groups. NS, normal saline; Bup, bupivacaine; Dexa, dexamethasone. Sale bar is at 10μm.

Fig 4. Caspase-3 expression in sciatic nerves.

4A. Immunohistochemical assays for cleaved caspase-3 staining (200×, nucleus counterstained with hematoxylin). There was no significant caspase expression in sciatic nerve axoplasm in all groups; 4B. Caspase-3 expression in positive control tissue (red arrows) (hippocampal neurons, 200×); 4C. Percentage of nucleus in sciatic nerves. The percentage of nucleus staining (blue arrows) in sciatic nerve was significant higher in bupivacaine treated mice on day 2 than day 7 (red box, *P<0.05), while no significant difference was found in other groups. 4D. Caspase-3 expression in mouse sciatic nerve after bupivacaine and high-dose dexamethasone block (400×). NS, normal saline; Bup, bupivacaine; Dexa, dexamethasone. Scale bar: 45 μm (black color),10 μm (red color).

Fig 5. S-100 expression in sciatic nerves.

5A. S-100 protein immunostaining in sciatic nerves (200×); 5B. Immunoreactivity of S-100 expression in different treatment groups on day 2 and day 7. Bupivacaine with or without intramuscular dexamethasone treatment group showed significantly lower S-100 expression on day 2 (red box) than that on day 7 (*P<0.05), while S-100 was expressed significantly higher when treated with perineural bupivacaine and high-dose dexamethasone than treated with perineural bupivacaine and low-dose of dexamethasone (^# P<0.05); 5C. Immunohistochemical assay for S-100 protein staining (deep brown, black arrows) in each group was primarily localized in the myelin sheath (400×, counterstained with hematoxylin). NS, normal saline; Bup, bupivacaine; Dexa, dexamethasone. Scale bar: 45 μm (black color),10 μm (red color).