PURPOSE: Botulinum toxin A have gained popularity and been used safely for chemodenervation of hyperfunctional facial lines. Manufacturers of the agent recommend storing the exotoxin at -5 C¢X or lower and using in 4 hours after reconstitution. Although freshly usage of the agent is highly recommended, there is no definite agreement in the literature. Some studies have shown that there is no decrease in potency of the toxin when stored after reconstitution; others revealed that refrigerated or refrozen toxin is not as potent as the fresh form. The aim of this study is to compare the ultrastructural alterations of the motor nerve and innervated muscle that appear following injection of freshly reconstituted and stored botulinum toxin.

MATERIALS AND METHODS: The study was carried out on 15 New Zealand white rabbits and anterior auricular muscle model was used for botulinum toxin injections. Three of the animals did not receive any injection (Group 1, control). Sterile saline was injected into the left anterior auricular muscles of three animals (Group 2, SALINE), right auricular muscles of all animals were used for botox injections. In Group 3 animals received an injection of 2.5 IU freshly reconstituted botox and sacrificed on the fifth day (Fresh botox early period, FBEP). In group 4 botulinum toxin which was reconstituted and stored 2 weeks at 4 C¢X in the refrigerator was injected and animals were sacrificed at fifth day (Stored botox early period, SBEP). In Group 5 freshly reconstituted botulinum was injected and animals were sacrificed at 12 weeks post injection (Fresh botox late period, FBLP). In Group 6 animals received stored botulinum injection and sacrificed at 12 weeks (Stored botox late period, SBLP). The muscle and the motor nerve were harvested at the end of waiting periods and examined for tissue degeneration using electron microscope. Amount of degeneration was scored using a semiquantitative method and analyzed statistically.

RESULTS: There were significant degenerative changes when muscles in the FBEP group (Group 3) were compared to the control group (Group 1) (p=0.037). When the FBLP muscles (Group 5) were compared to the control group (Group 1), sustained but improved atrophy was observed (p=0.046). When we compared the FBEP (Group 3) and SBEP (Group 4) muscles regarding atrophy parameters, muscles of both groups had severe degenerative findings and showed no significant difference (p>0.05). On the other hand, muscles of SBLP group (Group 5) had less severe degenerative findings than FBLP group (Group 6) muscles (p=0.043). On nerve evaluation, there were significant differences regarding the parameters of the tear and dehiscence in myeline sheath, axonal dispersion, degeneration in the Schwann cell cytoplasm, loss of non-myelinated nerves and degenerative observations in ground substance when the nerves in the FBEP group (Group 3) were compared to the control group (Group 1) (p=0,046). Similar but less severe results were observed when the nerves in the FBLP group (Group 5) were compared. When we compared the FBEP (Group 3) and SBEP (Group 4), nerves in FBEP group displayed more significant degenerative findings than the nerves in SBEP group (p=0.46). On the other hand, there was no significant difference in the severity of degeneration in the nerves in FBLP group comparing to the nerves in SBLP (pĨ0.05).

CONCLUSION: Alterations in muscle and nerve structures after botulinum toxin injection revealed that there is no significant difference between freshly reconstituted and stored toxin for 2 weeks, at the onset of the effect. However, when stored toxin was used atrophic changes in the muscle has started to return earlier or it is less severe than the fresh toxin, which may imply that fresh botulinum toxin is more durable than the stored toxin. On the other hand, fresh toxin displays an acute degenerative effect on the nerve, while stored toxin displays a slower onset. However, there is no significant difference between fresh and stored toxin at 3 months.