Ultrastructural evaluation of intramuscular applied botulinum toxin type A in striated muscles of rats

Botulinum toxin type A (BTX-A) is clinically utilized for therapeutic and cosmetic purposes in maxillofacial surgery as well as many other medical specialties. There is no sufficient ultrastructural research about BTX and it is controversial whether BTX-A causes muscle degeneration to some extent, in the course of therapy. The aim of this study was to evaluate the histological effects of BTX-A when injected into masseter and gluteal muscles.

A total of 30 male Sprague-Dawley rats were used and randomly divided into experimental (n =15) and control groups (n =15). Masseter and gluteal muscles were injected with a single dose of BTX-A in normal saline (0.5 U/0.1 ml), or 0.1 ml of normal saline, in the experimental and control groups, respectively. After 12 weeks all the rats were sacrificed. Gluteal, masseter muscles, and the sciatic nerves of the rats were prepared and electron microscopic, and light microscopic evaluation was performed on semi-thin sections cut from Epon embedded tissues and stained with toluidine blue. Quantitative parameters such as muscle fiber thickness and qualitative assessments including sarcosomal (striated muscle mitochondria) deformation, glycogen content, features of the triad structures and the intensity of connective tissue around the muscle fibers, and endoneurial and perineural tissue around nerve fibers were evaluated microscopically. We paired BTX- A (+) and BTX-A (-) samples statistically. Independent Samples t-test was used for the statistical analysis.

Muscle fiber's diameter was significantly decreased in BTX-A (+) group (p <0,001). Atrophic changes in the myofibrils were characterized by a decrease in the myofibrillar diameter and changes in the sarcomere structure, and were prominent in the BTX-A (+) group. Also, some other changes like dilatation in the sarcoplasmic reticulum cisternae, mitochondrial swelling, and clearing of mitochondrial cristae associated with degeneration, were detected. No morphologic difference in the sciatic nerve fibers was detected, and myelin sheaths of axon structures were intact in both groups.

BTX-A-induced muscular changes that are predominantly related to atrophy instead of degeneration. Although predominantly related to atrophy, our degeneration related findings suggest that further studies are needed focusing on detecting BTX-A effects on a cellular level. Hippokratia 2016, 20(4): 292-298.