This study aimed to evaluate whether oral zinc or copper supplementation affected the efficacy and sustainability of botulinum toxin-A (BTX-A) injection in masseter muscle of albino rats.

32 adult male albino rats were allocated equally into four groups: group I (control), group II received 10U BTX-A injection, group III received 10U BTX-A injection + zinc (1 mg, 4 days pre-injection), and group IV received 10U BTX-A injection + copper (0.04 mg, 7 days post-injection). Rats were euthanized at 2 and 12 weeks (4 rats per subgroup) after injection. The masseter muscle was examined via histological, histochemical, histomorphometrical and real-time polymerase chain reaction (qRT-PCR) analyses.

The histopathological results of the BTX-A group showed atrophied muscle fibers with increased atrophy with time compared to the control group. The BTX-A + zinc group displayed more atrophy compared to BTX-A group. Conversely, the BTX-A + copper group demonstrated improved histology of muscle fibers compared to BTX-A and BTX-A + zinc groups. Histomorphometric analysis of Masson trichrome staining at 2 and 12 weeks revealed that collagen area percentage was the highest in the BTX-A + copper and control groups, followed by BTX-A and BTX-A + zinc groups. At 12 weeks, the nuclear factor kappa beta (NF-κB) mRNA expression was significantly higher in BTX-A + zinc and BTX-A groups compared to BTX-A + copper group and relative to the control group.

Zinc supplementation significantly improved the effectiveness and durability of BTX-A, whereas copper supplementation reduced its efficacy.