Fresh and Stored Botulinum Toxin-A on Muscle and Nerve Ultrastructure: An
Electron Microscopic Study
Serhan Tuncer,MD, Suhan Ayhan,MD, Cıgdem Elmas,PhD, Deniz Erdogan,PhD, Engin
Calguner,PhD, Yavuz Basterzi,MD, Rabet Gozil,PhD, Meltem Bahcelioglu,MD
INTRODUCTION: Applications of botulinum toxin A for cosmetic reasons have gained
popularity and been used safely and effectively for chemodenervation of
hyperfunctional facial lines.1 The manufacturers of Botulinum toxin A recommend
storing the exotoxin at –5 C° or lower and diluting with physiologic saline
solution before application to obtain effective result. They also point that it
must be used in approximately 4 hours after reconstitution. Although freshly
usage of the agent is highly recommended, there is no definite agreement in
literature. Some studies have shown that there is no decrease in potency of the
toxin when stored after reconstitution;2 others revealed that refrigerated or
refrozen toxin is not as potent as the fresh form.3,4 These studies evaluated
neurophysiologic, microbiological and clinical changes related to stored fragile
toxin. However, there is no clear explanation about the ultrastructural
alterations following the injection of stored botulinum toxin.
In the present study, we aimed to compare the ultrastructural alterations on the
motor nerve and innervated muscle that appear following the injection of freshly
reconstituted and stored botulinum toxin.
METHOD: 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 and were used as the control group. In the
remaining 12 animals, sterile saline was injected into the left anterior
auricular muscle, and botulinum toxin A was injected into the right anterior
auricular muscle. Botulinum Toxin, Type A (Botox®, Allergan, Abdi İbrahim,
Turkey) which contains 100 units of neurotoxin was freshly reconstituted with 2
cc of sterile saline. Half of the prepared agent is used for fresh injection and
the remainder was stored at 4˚C in the refrigerator for two weeks. In all
animals, drooping ear was observed on the botulinum toxin injected side, which
indicated effective treatment. The animals were divided into 6 groups: 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° 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. Atrophy parameters, including location of the nucleus,
transverse striations and wiping out of Z lines, loss of myofibrils and
crystallization in mitochondria were examined to define muscle degeneration. On
the other hand, Wallerian degeneration parameters, including 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 were considered for nerve degeneration. The specimens were examined by
two separate histologists blindly and a semi-quantitative scoring system was used
to evaluate the amount of degeneration. If no degeneration criteria were observed
on histological examination “0” (zero) was given as the score and maximum
degenerative findings were scored with “5”. 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). (Figure 1)
A
B
C
D
Figure 1. (A) Fresh botulinum toxin early period. Note significant myofibriller
loss. (B) Fresh botulinum toxin late period. Minimal myofibriller loss and
degenerative Z bands. (C) Stored botulinum toxin early period. Loss of myofibril
and degeneration in Z bands. (D) Stored botulinum toxin late period. Striated
muscle with almost normal appearance
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
(p0.05). (Figure 2)
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 the
effects of fresh and stored toxin on nerve ultrastructure at 3 months.
A
B
C
D
Figure 2. (A) Fresh botulinum toxin early period. Note significant axonal and
myeline degeneration. (B) Fresh botulinum late period with stable axonal
degeneration. (C) Stored botulinum toxin early period. Significant axonal
degeneration. (D) Stored botulinum toxin late period. Nerve ultrastructure with
almost normal appearance
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