This study evaluated histological changes in masseter muscle fibres following reduced masticatory function by injection of botulinum toxin type A (BTX). Sixty 30-day-old Long-Evans male rats were randomly separated into four groups (15 per group): group I BTX masseter, 25 U/ml (0.04 ml each muscle) BTX was injected in bilateral masseter muscle whilst bilateral temporalis muscles received an equal amount of normal saline; group II BTX temporalis, 25 U/ml (0.04 ml each muscle) BTX was injected in bilateral temporalis muscle whilst bilateral masseter muscle received an equal amount of normal saline; group III BTX temporalis and masseter, bilateral temporalis and masseter were given 25 U/ml (0.04 ml each muscle) BTX; group IV normal saline (control), bilateral temporalis and masseter were given normal saline (0.04 ml each muscle). After 45 days, the rats were killed, the muscles dissected and mean muscle mass recorded. The superficial masseter muscles were immunohistochemically analysed. Fibre sizes in group III were bigger than those in other groups. There was a small percentage of type IIa fibres in group III. Reduction in muscle fibre size and transition of muscle fibre subtypes from type IIa to IIx or IIb fibres may occur due to reduced masticatory function.
According to the functional matrix theory introduced by M oss , changes in muscle function are followed by structural and morphological changes in the muscles themselves. There are evident impacts of these morphological changes in the maxilla and mandible on occlusion .
Much research on the impacts of masticatory function on craniofacial growth postulates that reduced masticatory function causes a reduction in craniofacial growth . Some studies indicate that reduced masticatory function can result in a reduced thickness of the cortical bone, decreased bone density, and delayed growth of the articulare . Histological changes in muscle fibres following reduced masticatory function are not well understood .
The methods most often used in previous studies to reduce masticatory function were changing the diet consistency and performing a myotomy or myoectomy , or denervating the muscle . The method of changing diet consistency reduces muscle function indirectly. Other methods cause scar tissue formation and affect growth. The true effects of atrophy of masticatory muscles on craniofacial growth largely remain unknown.
In recent years, botulinum toxin type A (BTX) has been widely used. After injection into muscle tissues, BTX binds to nerve endings in neuromuscular junctions and inhibits the release of acetylcholine. Muscular contracture is inhibited, and muscle function is reduced . BTX can reduce muscle function without major tissue damage. If BTX is incorporated to reduce masticatory function, the effects of atrophy of the masticatory muscles on craniofacial growth can be better understood.
The purpose of this study was to clarify changes in masseter muscle fibres due to BTX injections into growing masseter and/or temporalis muscles to reduce masticatory function. Interactions of masseter and temporalis muscles, which play the main roles in masticatory function, are also discussed.
Materials and methods
Sixty 30-day-old male Long-Evans rats were randomly separated into four groups of 15 rats: group I BTX masseter, 25 U/ml (0.04 ml each muscle) BTX was injected in bilateral masseter muscle whilst bilateral temporalis muscle was given an equal amount of normal saline; group II BTX temporalis, 25 U/ml (0.04 ml each muscle) BTX was injected in bilateral temporalis muscle whilst bilateral masseter muscle was given an equal amount of normal saline; group III BTX temporalis and masseter, bilateral temporalis and masseter were given 25 U/ml (0.04 ml each muscle) BTX; group IV normal saline (control), bilateral temporalis and masseter were given normal saline (0.04 ml each muscle) ( Table 1 ).
|Masseter muscle||Temporalis muscle|
|Group I BTX masseter||BTX *||Normal saline **|
|Group II BTX temporalis||Normal saline||BTX|
|Group III BTX temporalis and masseter||BTX||BTX|
|Group IV normal saline (control) temporalis and masseter||Normal saline||Normal saline|
Masseter muscle injections consisted of injections into both the superficial and deep masseter muscle layers. The superficial masseter muscle injection site was a point halfway along a line intersecting the orbitale and outer meatus, perpendicular to a line joining the mandibular plane angle and outer oral commissure. The deep masseter muscle injection site was a point immediately posterior to the orbitale on a line connecting the outer meatus and orbitale, perpendicular to a line joining the mandibular plane angle and outer oral commissure. The injection sites for the temporalis muscles were located one-third and two-thirds along a line connecting the orbitale and the outer meatus.
After 45 days, the 75-day-old rats were perfused with 4% formaldehyde and killed. The masseter and temporalis muscle were dissected carefully and harvested by the same operator. After the right masseter and temporalis muscles were dissected, they were weighed with a precision balance (model ZSA80, Scientech, Denver, CO, USA). The master muscles were immersed in 4% formaldehyde for 7 days and divided into superficial and deep portions. Cuts were obtained only from the middle portion of the belly of the superficial masseter muscles perpendicular to the main orientation of the muscle fibres. An immunohistochemical analysis of the superficial masseter muscles was performed.
The masseter muscles of rats consist of only type II muscle fibres , which are categorized into three subtypes (types IIa, IIx, and IIb). Type IIa fibres can be identified with a monoclonal antibody (mAb) to myosin (A4.74), and type IIx fibres can be identified with a mAb to myosin (MYH1). The remaining percentage (apart from type IIa and IIx muscle fibres) indicated type IIb muscle fibres. The experimental protocol was approved by the Laboratory Animal Research Committee of Taipei Medical University, Taipei, Taiwan.
Samples were observed with Image-Pro Plus ® image software. All samples were digitized twice by the same technician. The size of the muscle fibres and the percentages of muscle fibre subtypes in the four groups were calculated.
All data were analysed by one-way ANOVA followed by the Tukey’s honestly significant difference (HSD) test. Tukey’s HSD test is a single-step multiple comparison procedure and statistical test generally used in conjunction with an ANOVA to find which means are significantly different from one another. It compares all possible pairs of means.
During the experimental period, the overall body weights of rats steadily increased. There were no significant differences in weight gain between the four groups during the experimental period ( Fig. 1 ). Injections of BTX did not interfere with the overall growth of the rats.
The weights of the masseter muscles in groups I, II, and IV were significantly greater than that in group III, and the weight of the masseter muscles in group IV was significantly greater than that in group II ( Fig. 2 ).
The weights of the temporalis muscle in groups I and IV showed no significant difference, and were significantly greater than that in group II, which was significantly greater than that in group III ( Fig. 3 ).
Muscle fibres in groups I, II, and IV were significantly smaller than those of group III, with a mean ranking of IV > I > II > III. Differences amongst mean muscle fibre sizes were significant between groups I and III, II and III, and IV and III but not amongst groups I, II, and IV ( Fig. 4 ).
The results of immunohistochemical analysis are shown in Figs 5 and 6 . Group III contained significantly fewer type IIa fibres than group II. But there were no significant difference amongst the percentages of type IIx and IIb fibres in all groups ( Figs 7–9 ).