THE AMERICAN JOURNAL OF ANATOMY i~(9:93-110ii99o)

Craniofacial Alterations Following Electrolytic Lesions of the Trigeminal Motor Nucleus in Actively Growing Rats KENNETH E. BYRD, STEPHEN T. STEIN, ALAN J. SOKOLOFF, ANT) KAVITA SHANKAR Department of Basic Sciences (K.E.B., A.J.S.,K.S.1, University o f Southern California School o f Dentistry, Lns Angeles, California 90089; University of California at Sun Francisco School of Dentistry, Sun Francisco, California 94143 (S.T.S.)

ABSTRACT The objective of this study was to define further the role of the trigeminal motor nucleus (TMNu) in the postnatal ontogeny of the mammalian craniofacial skeleton. To that end, 42 male Sprague-Dawley rats underwent stereotaxic surgery at 40 days of age; 21 received small electrolytic lesions to their left-side TMNu (lesioned group) while 21 had TMNu stimulation with no actual electrolytic lesion produced (sham group). Seven rats from each group were killed at 28, 56, and 84 days postoperative to analyze trigeminal motoneuron (TMNe) count, masticatory muscle weight, and osteological growth vector data. At all three time periods, lesioned animals showed significant differences 1) between the surgery and nonsurgery sides, and 2) from sham animals. However, sham animals also demonstrated significant between-side differences for medial pterygoid muscle weight (56 days), mandibular height (28 and 56 days), and mandibular length data (84 days); these data suggested that even relatively slight damage to TMNe can create morphological changes within the craniofacial complex. Snout deviation in a lesioned rat towards the opposite side from all other lesioned animals was correlated with unique damage to its pontine reticular formation; this suggested that the observed morphological alterations of the craniofacial complex may have been due not only to TMNu damage, but also to changed expressions of the masticatory central pattern generator (CPG). Morphological alterations of the craniofacial skeleton resulting from lesions to the TMNu were likely due to changed neuromuscular activity patterns of the masticatory muscles and their biomechanical effects upon bone.

ticatory) forces due to soft diets effect a reduction in bone growth and remodeling in the craniofacial complex (Beecher and Corruccini, 1981; Bouvier and Hylander, 1981, 1982, 1984; Bouvier and Zimny, 1987). Altered biomechanical forces t h a t directly effect skeletal asymmetries of the craniofacial complex can be produced by lesioning motoneurons within the brainstem (Gardner et al., 1980; Phillips e t al., 1982; Byrd, 1984,1988a).This technique essentially obviates the confounding effects of sensory loss, tissue-scarring, and circulatory interruptions due to nerve and muscle resection methods (Johnston, 1976). Recent data from our laboratory have demonstrated that lesions to the facial and trigeminal motor nuclei of growing rats effect 1)atrophy of lesion-side muscles of facial expression and mastication, 2) asymmetry of zygomatic arch and dorsal face skeletal dimensions, 3) malocclusions, 4) hemimandibular microsomia, and 5) progressive reduction of the mandibular condyle (Byrd, 1988a). We were unable at that time to determine the relative contributions of the facial and trigeminal neuromuscular complexes (FNC and TNC) to craniofacial growth and development. The purpose of this study is to define further the role of the trigeminal motor nucleus (TMNu) in the postnatal development of the mammalian craniofacial skeleton. These data, derived from actively growing rats, may serve as a n animal model for the morphological sequelae (muscular and skeletal asymmetries) seen in humans with hemifacial microsomia (Pruzansky, 1969; Vargervik and Miller, 1984) and plagiocephaly (Kreiborg et al., 1985). Detailed electromyographic and mandibular movement data (as in Byrd, 1984, 1988b,c) from lesioned animals in this study are currently being analyzed and will be published elsewhere. MATERIALS AND METHODS Experimental Animals

A total of 42 male Sprague-Dawley strain rats ( R a t t u s norvegicus) were used in this study and under-

INTRODUCTION

Neuromuscular influences upon skull growth and development, although initially described by Wolff (1892) in his “Law of Transformation,” received little experimental verification until after the Second World War (Washburn, 1946, 1947; Horowitz and Shapiro, 1951, 1955; Avis, 1959, 1961). These early studies involved resections of nerves andlor muscles in order to document effects of facial and masticatory muscles upon craniofacial form. Recently, it has also been demonstrated that decreased biomechanical (reduced mas-

c) 1990 WILEY-LISY. INC

went stereotaxic surgery a t 40 days of age (160 gm). All rats were housed individually in steel cages (20.3 cm high x 38.1 cm wide x 55.9 cm deep) at a constant temperature of 76°F; all animals were kept within the quiet confines of the University of Southern California vivarium before and after surgical procedures. Ambi-

Received December 21, 1989. Accepted May 21, 1990.

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TABLE 1. Measurements (see Fig. 1). Paired cranial measurements 1. Midline frontonasal suture to premaxillonasal suture (E-I, E-I’j 2. Maxillozygomatic suture to premaxillonasal suture ((2-1, C’-I’j 3. Zygomaticosquamosal suture to premaxillofrontomaxillary suture (D-J, D‘-J‘) 4. Zygomaticosquamosal suture to midline frontonasal suture (D-E, D‘-E) 5 . Anteriormost incisopremaxillary contact to anterior first molar (K-M, K’-M’) 6. Anteriormost incisopremaxillary contact to occipitobullobasisphenoid contact (K-R, K‘-R’) 7. Premaxillomaxillary suture to anterior first molar (P-M, P’-M‘) 8. Premaxillomaxillary suture to maxillozygomatic suture (P-C, P‘-C‘) 9. Anterior first molar to maxillozygomatic suture (M-C, M’-C‘) 10. Anterior first molar to zygomaticosquamosal suture (M-D, M’-D‘) 11. Posterior third molar to maxillozygomatic suture (N-C, N’-C‘) 12. Posterior third molar to zygomaticosquamosal suture (N-D, N‘-D‘j 13. Posterior third molar to occipitobullobasiphenoid contact (N-R, N‘-R‘) 14. Posterior palatal spine to maxillozygomatic suture (Q-C, Q-C‘) 15. Posterior palatal spine to occipitobullobasiphenoidcontact (Q-R, Q-R’) 16. Occipitobullobasiphenoid contact to maxillozygomatic suture (R-C, R’-C‘) Paired mandibular measurements 17. Gonial angle to superiormost incisorcorpus contact (S-T, S’-T’) 18. Gonial angle to posterior third molar (S-W, S’-W’) 19. Gonial angle to superiormost coronoid process (S-Y, S’-Y‘) 20. Gonial angle to anteriormost condylar articular surface (S-Z, S’-Z‘) 21. Superiormost incisorcorpus contact to posterior third molar (T-W, T’-W’j 22. Superiormost incisorcorpus contact to superiormost coronoid process (T-Y, T‘-Y’j 23. Superiormost incisorcorpus contact to anteriormost condylar articular surface (T-Z, T’-Z’j 24. Inferiormost incisorcorpus contact to superiormost coronoid process (U-Y, U’-Y7 25. Inferiormost incisorcorpus contact to anteriormost condylar articular surface (U-Z, U’-Z‘) 26. Anterior first molar to superiormost coronoid process (V-Y, V‘-Y’j 27. Posterior third molar to superiormost coronoid process (W-Y, W’-Y‘) 28. Posterior third molar to anteriormost condylar articular surface (W-Z, W’-Z’) 29. Superiormost coronoid process to anteriormost condylar articular surface (Y-Z, Y’-Z’) 30. Maximum mandibular height (parallel planes between inferior mandible and point Y) 31. Maximum condylar width (lateralmost and medialmost articular surfaces) 32. Maximum condylar length (anteriormost and posteriormost articular surfaces) Unpaired cranial measurements 33. Maximum length (A-B) 34. Anterior zygomatic arch width (C-C’) 35. Posterior zygomatic arch width (D-D’) 36. Binasal width (1-1‘) 37. Bifacial width (J-J’) 38. Bipremaxillary width (P-P’) 39. Anterior bimolar width (M-M’) 40. Posterior bimolar width (N-N’) 41. Bitympanic width (0-0’) 42. Occipitobasisphenoid width (R-R’) 43. Maximum height (hard palate-calvarial

vault)

Unpaired mandibular measurements 44. Maximum width (S-S‘) 45. Bicoronoid width (Y-Y’) 46. Bicondvlar width (Z-2’)

ent lighting conditions were controlled with alternating periods of artificial light and darkness: LD 12:12 with light from 0630 to 1830 hr; normal light intensity was maintained by the USC vivarium staff. All rats were maintained on a diet of standard rat pellets (Purina); postsurgically, all rats were given watersoftened and powdered pellets ad libitum in addition to the hard pellets. All animals were maintained in the above conditions after weaning a t 23 days of age except when they were removed for stereotaxic surgery and recovery (two days). General anesthesia was administered by intramuscular injection (lateral thigh) of the following mix-

ture: ketamine 100 mg/ml, rompun 20 mg/ml, and acepromazine 10’mgiml in the amount of 0.01 cc per 10 gm of body weight. In addition, 0.02 cc atropine sulfate (0.54 mg/ml) and 0.40 cc chloramphenicol sodium succinate (100 mg/ml) were injected intraperitoneally after each animal was sedated to prevent aspiration during surgery and postoperative infections. All animals were also given a n oral suspension of chloromycetin palmitate (1.0 ml) daily for one week postoperatively as a n infection prophylaxis. All 42 stereotaxic surgeries were performed between November 13, 1987, and July 23, 1988, typically between 1000 and 1800 hr PST on these dates. All sur-

CRANIOFACIAL (:HANGES AFTER MOTOR V 1,E:SIONS

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Fig. 2. Malnccluded incisors of‘lesioned animal a t 20 days postoperative. The photograph was taken of an anesthesized animal prior to incisor reduction with a dental handpiece, necessary because of incisor hypereruption (see text). Notice nonsurgery-side (right)deviation of 1) the bases of maxillary incisors, 2) maxillary gingivae, and 3) nose.

Y

A

I ?

& -S

w UY

z \

S-

“\

w

u

Flg. 1. Osteometric points used in this study (after Byrd, 1988a). Primed (‘) letters denote right (nonsurgery) side measurement points. For description, refer to Table I and text. (Reproduced with the permissinn of‘ Alan R. Liss, Inc).

gical animal-care procedures were fully in compliance with the “Guide for the Care and Use of Laboratory Animals” (DHEW Publication No. (NIH) 73-23). The 42 rats were divided into experimental (lesioned) and control (sham-lesioned) groups of 21 each. Although both groups had stereotaxic surgery at 40 days of age, experimentals received a small electrolytic lesion in their left-side TMNu, while controls received a sham lesion (TMNu stimulation with no actual electrolytic lesion produced). Seven rats from each group were killed a t 28, 56, and 84 days postoperative. Stereotaxic Surgery Procedure

Surgical protocol was essentially identical to that dcscribed in earlier studies (Gardner et al., 1980; Byrd,

1984, 1988a): a midline incision was made over the sagittal suture, integument and periosteum were reflected, and a 4-mm diameter hole was drilled through the bone in order to expose the dura mater. Both lesioned and sham animals had a hole drilled directly over their left-side TMNu according to the stereotactic coordinates given by Paxinos and Watson (1982). Exposed tissues were liberally irrigated by solution of chloramphenicol sodium succinate (100 mg/ml) in order to prevent infection during the surgery. Sterile surgical procedures were used for each of the 42 surgeries. Experimentalilesioned animals had unilateral electrolytic lesions (1.5 mA, 15 sec) placed in their left-side TMNu in the manner documented by Byrd (1984, 1988a) and using a Kopf model 900 stereotaxic unit. After piercing the dura with a 27-gauge, sterile hypodermic needle, a Rhodes NEX-100, concentric, bipolar electrode was slowly lowered while continuously stimulating the TMNu a t 1 pulse per sec at between 2 and 0.1 V. The electrode descended in a vertical axis, usually 1.9 mm to the left and 0.2 mm posterior to stereotaxic earbar zero. Actual electrolytic lesions were produced in the TMNu after a nucleus injury response (NIR) was observed. NIRs are caused by mechanical trauma to TMNu motoneurons by a n electrode passing through them (Byrd, 1984). In rats, a NIR can take the form of 1) non-rhythmic mandibular movements, 2) sudden mandibular depression or protraction, 3 ) a continuous fasciculation (“worming”) of the stimulationside masticatory muscles, or 4)some combination of the above. Once a n NIR was detected, the electrode was lowered a n additional 0.5 mrn and the actual electrolytic lesion (1.5 mA, 15 sec) produced with a Grass direct-current lesion marker (Byrd, 1984, 1988a). Sham-lesioned animals underwent identical surgical procedures, including having their TMNu stimulated

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Fig. 3. Cresyl-violet sections of representative sham ( S )and lesioned (L) animals killed at 56 and 84 days postoperative. Large arrows denote nonsurgery-side (right) and small arrows indicate surgery-side (left) TMNu. Notice fewer trigeminal motoneurons in surgery-side

TMNu of lesioned animals compared to shams. A reduction in the number of' neurons within the surgery-side mesencephalic trigeminal (Mes-V)nuclei is also apparent for the lesioned animals.

until a NIR was detected, but no actual electrolytic lesion was produced. Both lesioned and sham animals then had their wounds closed (5.0 silk sutures for periosteum, 3.0 silk sutures for integument), treated with betadine, cleaned, and coated with fingernail polish to prevent them from scratching open their sutures.

out intact and weighed to the nearest 0.01 gm with a n electronic Ohaus scale. After dissection, each muscle was stored in 70% ethanol and blotted between 5 cm x 5 cm gauze squares under a 500 gm weight for 5 sec just before weighing. The left- and right-side anterior digastric, posterior digastric, lateral pterygoid, medial pterygoid, masseteric complex (the combined superficial, deep, maxillomandibularis, and zygomaticomandibularis portions; Turnbull, 19701, and temporalis muscles of the 21 lesioned and 21 sham rats were all dissected out intact and weighed for this study. After brainstem removal and muscle dissection, the remaining tissue was cleaned off; and dry skull preparations were made of the 42 animals. A combined total of 46 cranial and mandibular measurements were then taken from both lesioned and sham groups (see Table 1 and Fig. 1)to the nearest 0.1 mm with a Helios needlepoint dial caliper in order to provide accurate growth vector data. Detailed descriptions of the osteometric points S, Y, and Z and the measurements of maximum mandibular height, maximum condylar length, and maximum condylar width are given in Byrd (1988a). Cranial measurements involving the osteometric points F, G , and H were not taken because of posterior cranial vault destruction during brainstem removal for histological analyses.

Data Collecfionand Analyses Lesioned and sham rats (7 each) were killed a t 28, 56, and 84 days postoperative. Transcardiac perfusion with physiological saline (0.9% solution) followed by 10% buffered formalin occurred a t that time. The brainstem of each animal was then removed for histological documentation of lesion extent. Paraffin embedding of the pons occurred. After tissue stabilization in 70%)ethanol, the pons was embedded in Ameraffn; and 10 Fm sections were cut, mounted, and stained with cresyl-violet (LaBossiere and Glickstein, 1976). Trigeminal motoneuron (TMNe) counts were then made for the TMNu of all 42 animals by one of us (AJS) using a Nikon Labophot microscope and JAVA cell-imaging system. The surgery- and nonsurgery-side TMNu's of 4,079 10-pm sections were analyzed in this study. Only TMNe with a definite nucleolus and exhibiting no signs of chromatolysis were counted. The muscles of mastication were carefully dissected

CRANIOFACIAL CHANGES AFTER MOTOR V 1,ESIONS

TABLE 2. Trigeminal motoneuron counts (X Days postoperative 28 66

k

S.D.)

Lesioned Shams Left Right Left Right 1,115.43 5 75.21 ' 495.14 5 460.02 1,205.14 -c 79.27"'k 1,088.71 t 93.47+ (n = 7 ) (n 7) (n = 7) (n = 7) 570.29 i 205.94 1,152.14 5 80.57*' 1,132.14 t 102.42.' + 1,129.29 3- 70.91 (n = 7) (n = 7) (n = 7 ) (n = 7) 324.29 t 292.60 1,109.14 72.49*" 1,106.14 rt 116.6Gc ' 1,123.43 2 95.97 (n = 7) (n = 7 ) (n = 7) (n = 7)

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Craniofacial alterations following electrolytic lesions of the trigeminal motor nucleus in actively growing rats.

The objective of this study was to define further the role of the trigeminal motor nucleus (TMNu) in the postnatal ontogeny of the mammalian craniofac...
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