2.3. Research procedure

BALB/c mice were considered healthy if they were actively moving, had bright eyes, did not have dull hair, and had no defects. The dose of mg/kg weight of lidocaine was converted to volume in units of ml. The sterile distilled water solution was given in ml according to the volume of lidocaine. The expression of mRNA NF-kβ was assessed in units of fold change [28]. The protein levels of NF-kβ are expressed in picograms (pg/ml). The protein levels of TNF-α are expressed in nanograms (ng/ml).

The treatment group. After 4h of experiencing a musculoskeletal injury, they were given an intravenous injection of lidocaine (Kimia Farma Ltd, Jakarta, Indonesia) 2 mg/kg body weight through the tail vein, once every 2h, continuously for 24h. The milligram dose of lidocaine was converted to volume in milliliters (ml).

The control group. After 4h of suffering from sterile musculoskeletal injuries, they were given an intravenous injection of sterile distilled water through the tail vein instead of lidocaine injection.

Before inducing sterile musculoskeletal injuries, 0.3 ml of mice blood was taken from the tail vein and mixed with L6 buffer solution (first blood test). The mice were then anesthetized by injecting ketamine (Combiphar Ltd, Jakarta, Indonesia) 50 mg/kg intraperitoneally. After the mice were sedated, the left thigh was shaved clean. The musculoskeletal injury was performed in a sterile manner. The groin of the mice was clamped firmly with one needle holder for fixation, and the middle of the femur was also clamped with the other needle holders. The left thighbone of the mouse was broken by moving the needle holders located in the middle of the thigh up and down against the needle holders in the groin until a cracking sound was heard and the fracture of the bone in the thigh when moved by hand was felt [29]. It was stabilized with external fixation and the mice were then returned to their cages. Metamizole 50 mg/kg orally was given as an analgesic for two consecutive days. After 4h of suffering from sterile musculoskeletal injuries, 0.3 ml of blood was taken again through the tail vein (second blood test).

The lidocaine group mice were then given an injection of lidocaine at a dose of 2 mg/kg BW through the tail vein, once every 2h for 24h, while the control group was given an injection of sterile distilled water as a substitute for lidocaine. Two hours after the injection of lidocaine and sterile distilled water was completed, 0.3 ml of mice blood was taken again through the tail vein (third blood test). A fourth and final blood sample was drawn at 24h (fourth blood test). Mice were euthanized by cervical dislocation under anesthesia 2 days after musculoskeletal injuries.