Materials and Methods

This was an open-label study over 7 weeks. Prior to the start of the study, full protocol and ethics approval were obtained from the animal centre of Taipei Medical University. 60 male growing Long-Evans rats, weighing approximately 120 g, were enrolled in the study. All study animals were male to limit sexual dimorphism. Study animals were housed in separate cages in the same room under light and climate-controlled conditions, and were fed a standard diet of hard pellets and water ad libitum . Mean weights (accuracy to nearest 0.01 g) were measured during the 7-week experimental period (T1–T7) with an electronic digital scale.

At age 4 weeks (T0), weaned study animals were injected with BoNT/A (Botox ^® , Allergan Pharmaceuticals, Irvine, CA, USA). BoNT/A was diluted with 4.0 ml sterile, non-preserved 0.9% saline to yield 25 U/ml preparation. Study animals received a 2.5-ml intramuscular injection of BoNT/A (1.0 U/muscle mass) as follows. Group I (Mb + Tns): injection into bilateral masseter muscles (bilateral temporalis muscles received equivalent amounts of 0.9% sterile, non-preserved saline). Group II (Mns + Tb): injection into bilateral temporalis muscles (bilateral masseter muscles received equivalent amounts of 0.9% sterile, non-preserved saline). Group III (Mb + Tb): injection into bilateral masseter and temporalis muscles. Group IV (control, Mns + Tns): injection of 0.9% sterile, non-preserved saline into bilateral masseter and temporalis muscles.

A single dose of 50 mg/kg ketamine (Zoletil ^® , Virbac Inc., CA, USA) and 10 mg/kg xylazine (Rompum ^® , Bayer Inc., Toronto, Canada) were administered intraperitoneally to achieve initial anaesthesia for injections of BoNT/A and saline. The bilateral injection sites for the temporalis muscles were located one-third and two-thirds along the line connecting the orbitale and outer meatus. Masseter muscles received superficial and deep muscle layer injections. The superficial muscle site was located halfway along the line connecting the orbitale and outer meatus, perpendicular to the line adjoining the mandibular plane angle and outer oral commisure. The deep muscle site was posterior to the orbitale on the line connecting the outer meatus and orbitale. Every temporalis injection site received 1.0 U and every masseter injection site received 0.5 U of BoNT/A or the equivalent amount of saline. At the end of the 7-week experimental period, the rats were perfused and killed.

The masseter and temporalis muscles were dissected carefully and harvested by the same operator (W.C.C.). The mean muscle mass was recorded with a precision balance (model ZSA80, Scientech, Denver, USA) and differences were compared among Groups I, II, III and the control. Dried skulls were prepared for direct cranial, maxillary and mandibular anthropometric measurements. 40 anthropometric parameters were analyzed ( Fig. 1 a and b ) as described by L evrini et al. , Y amamoto , U lgen et al. , T sai and L iao , and M atic et al. .

(a) Landmarks of anthropometric points. Cranial anthropometric points: 1, Internasal point; 2, Nasofrontal point; 3, Lateral nasal point; 4, Orbita point; 5, Zygion point; 6, Frontoparietal point; 7, Squama temporalis point; 8, Occipital point; 9, Tympanic point; 10, Nasomaxillary point. Maxillary anthropometric points: 11, Key ridge point; 12, Zygomatic arc, inferior point; 13, Zygomatic arc, anterior point; 14, Incisive foramen, lateral point; 15, Posterior nasal spine; 16, Prosthion point; 17, Incisive superior alveolar point; 18, U1, incisal point; 19, U1, cervicolabial point; 20, U1, cervicopalatal point; 21, U1, mesial point; 22, U1, distal point; 23, U6, mesial point; 24, U6, mesiobuccal cusp point; 25, U8, distal point; 26, U8, mesiobuccal cusp point. Mandibular anthropometric points: 27, Coronoid point; 28, Coronoid notch point; 29, Condylion point; 30, Gonion point; 31, Gnathion; 32, Mn point; 33, Antegonial incisure; 34, Anterior masseteric line; 35, Menthon point; 36, Mandibular alveolar point; 37, Infradental point; 38, Incisive inferior alveolar point; 39, L1, incisal point; 40, L1, cervicolabial point; 41, L1, cervicolingual point; 42, L1, mesial point; 43, L1, distal point; 44, L6, mesial point; 45, L6, mesiobuccal cusp point; 46, L8, distal point; 47, L8, mesiobuccal cusp point. (b) Anthropometric measurements. Cranial Measurements: 1, Total skull length; 2, Nasal length; 3, Nasal width; 4, Interorbital width; 5, Interzygomatic width; 6, Maximum skull width; 7, Maximum skull height; 8, Upper anterior facial height; 9, Lower anterior facial height; 10, Total anterior facial height. Maxillary measurements: 11, Total maxillary length; 12, Incisive foramen width; 13, Maxillary width; 14, U6 intermolar width; 15, U8 intermolar width; 16, U1 incisor crown height; 17, U6 height; 18, Maxillary posterior arch length; 19, U1, labiolingual distance; 20, U1, mesiodistal distance. Mandibular measurements: 21, Mandibular length I; 22, Mandibular length II; 23, Mandibular length III; 24, Corpus length; 25, Ramus height I; 26, Ramus height II; 27, Ramus height III; 28, Ramus height IV; 29, Corpus height; 30, Mandibular plane angle; 31, Bicoronoidal width; 32, Bicondylar width; 33, Bigonial width; 34, L6 intermolar width; 35, L8 intermolar width; 36, L1, incisor crown height; 37, L6 height; 38, Mandibular posterior arch length; 39, L1, labiolingual distance; 40, L1, mesiodistal distance.

Animal weight data were compared using repeated measures of analysis of variance (ANOVA) followed by post hoc tests using least square deviation. For description of data, mean values and standard deviations were calculated and presented as mean ± SD. Changes in muscle mass and anthropometric measurements were analyzed using one-way ANOVA followed by the Mann–Whitney U -test for comparison between experimental groups and control. Statistical significance was defined at the 5% level. All statistical analyses were performed using SPSS version 13.0 software (SPSS Inc., Chicago, IL, USA).

Results