Morphological and Histological Changes Following Triamcinolone Injection Alone or Mixed Injection of Triamcinolone and Botulinum Toxin in Upper Eyelid
Authors: Vu Que Anh , Kwang-Eon Choi , Thai Duy Quang , Baek Sehyun 
Published: November 2024

Objective:
The purpose of this experiment was to evaluate the functional and histologic changes in the upper eyelid muscles after injection of triamcinolone acetonide (TA) alone or TA combined with botulinum toxin A (Botox or BTXA) in the cynomolgus monkey model.

Methods:
Twenty eyes of 10 cynomolgus monkeys were divided into 4 groups: 3 experimental groups (1, 2, and 3) and the control group (group 4) based on the injection type. In group 1, 0.5 mL of TA (Kenalog 40 mg/mL) was administered subconjunctivally (between the conjunctiva and the Müller muscle) with a 26 G needle to the inverted upper eyelid of one eye. In group 2, the same procedure was done with 0.5 mL TA injected into the other upper eyelid. After that, 5 UI/0.1 mL Botox was injected transcutaneously into the suborbicularis oculi space in the middle third of the upper eyelid 3 mm above the center of the superior tarsal border. In group 3, a subconjunctival of 0.5 mL TA was administered to the inverted upper eyelid of one eye 3 times: the injection day, 2 weeks, and 6 weeks after the first injection, whereas a normal saline injection of the same volume was administered one time to the other eye at the injection day in group 4 (control group). Follow-up was done to evaluate the clinical changes in eyelid position at 2, 6, and 12 weeks after injection. Hematoxylin-eosin and Masson trichrome were used to assess the levator or Muller muscle histology and measure the fiber diameter.

Results:
During the clinical follow-up, there were no major complications observed in any monkeys. The macroscopic appearance of the upper lid on the biopsy day did not differ among groups 1, 2, and 4. There were no cases in these groups that had subconjunctival TA deposit 3 months after injection. Conversely, in group 3, there were 3/5 eyes showing the subconjunctival TA deposit at the last examination.
No specific changes in the marginal reflex distance 1, marginal reflex distance 2 (MRD2), and lid crease were noted in either TA alone injection groups 1, 3, and 4. In contrast, there was a significant decrease in marginal reflex distance 1 at 2 weeks (P = 0.003) and 6 weeks (P = 0.005) after TA injection in group 2 in comparison to the baseline.
In terms of MRD2, while in group 2, there were significant differences between the pre-MRD2 and the post-MRD2 till the 2 weeks after injection (P = 0.006), then it became insignificant from the sixth week afterward. In contrast, at 2 weeks after injection, MRD2 was reduced in both TA-injected groups 1 and 3, but the observed difference was not significant in both groups. At 6 and 12 weeks, MRD2 fluctuation was not remarkable in these 2 groups and there were no significant differences in comparison to the baseline (P > 0.05).
Histological evaluation showed that Müller muscle does not attach directly to the superior border of the tarsus, but it changes to the tendon before attaching to the tarsal plate. In addition, there were no statistical differences in levator muscle fiber diameter and Müller muscle fiber diameter between the 4 groups, with P = 0. 621 and P = 0.695, respectively.

Conclusion:
Triamcinolone acetonide combined with BTXA showed better results in decreasing upper eyelid height than TA alone in normal monkey eyelids due to its predictable effect. In addition, there were no differences between the side effects and the histology results between the 4 groups. Therefore, TA combined with BTXA may become a promising treatment for selective thyroid eyelid retraction and could offer an alternative to surgery and its complications.