Inhibiting Matrix Metalloproteinases Protects Evoked Electromyography Amplitudes and Muscle Tension in the Orbicularis Oris Muscle in a Rat Model of Facial Nerve Injury.

Facial nerve injury results in degradation of the neuromuscular junction (NMJ) and blocks neurotransmission between the pre- and postsynaptic structures, which are separated by a synaptic cleft. Matrix metalloproteinases (MMPs), enzymes that degrade and modify the extracellular matrix, play critical roles in regulating NMJ remodeling. We previously demonstrated that MMP1, MMP2, MMP3, MMP7, and MMP9 are overexpressed in facial nerve-innervated orbicularis oris muscle after facial nerve injury in a rat model. In the present study, the MMP inhibitor prinomastat was administered to rats after facial nerve injury. The MMP levels, agrin expression, and muscle-specific kinase (MuSK) phosphorylation were evaluated. Variations in evoked electromyography (EEMG) amplitude were also recorded. Compared with the control group, MMP expression in the orbicularis oris after facial nerve injury was significantly reduced in the prinomastat group. Inhibition of MMP expression maintained agrin expression and MuSK phosphorylation; the NMJ morphology was also protected after the injury. Moreover, prinomastat treatment sustained EEMG amplitude and muscle tension after the injury. These findings indicate that inhibiting MMPs can protect the function and morphology of the NMJ and demonstrate the need for protection of the NMJ at early stages after facial nerve injury.

In the human, true myocutaneous junctions of skeletal muscle fibers are limited to the face.

Within the panniculus carnosus-associated skeletal muscles in the human, the palmaris brevis and the platysma showed myotendinous/myofascial junctions with clear distance to the corium and the specific connection collagen type XXII. The orbicularis oris muscle, in contrast, contained bundles of striated muscle fibers reaching the corium at two distinct levels: the predominant inner ending was connected to the elastic network of the inner corium and the outer ending was within the more superficial collagen network. At both locations, the striated muscle fibers showed brush-like cytoplasmic protrusions connecting a network which was not oriented toward the muscle fibers. Collagen type XXII was not present.

Interference with SRF expression in skeletal muscles reduces peripheral nerve regeneration in mice.

Traumatic injury of peripheral nerves typically also damages nerve surrounding tissue including muscles. Hence, molecular and cellular interactions of neighboring damaged tissues might be decisive for successful axonal regeneration of injured nerves. So far, the contribution of muscles and muscle-derived molecules to peripheral nerve regeneration has only poorly been studied. Herein, we conditionally ablated SRF (serum response factor), an important myofiber transcription factor, in skeletal muscles of mice. Subsequently, the impact of this myofiber-restricted SRF deletion on peripheral nerve regeneration, i.e. facial nerve injury was analyzed. Quantification of facial nerve regeneration by retrograde tracer transport, inspection of neuromuscular junctions (NMJs) and recovery of whisker movement revealed reduced axonal regeneration upon muscle specific Srf deletion. In contrast, responses in brainstem facial motor neuron cell bodies such as regeneration-associated gene (RAG) induction of Atf3, synaptic stripping and neuroinflammation were not overly affected by SRF deficiency. Mechanistically, SRF in myofibers appears to stimulate nerve regeneration through regulation of muscular satellite cell (SC) proliferation. In summary, our data suggest a role of muscle cells and SRF expression within muscles for regeneration of injured peripheral nerves.

Two Pathways Regulate Differential Expression of nAChRs Between the Orbicularis Oris and Gastrocnemius.

BACKGROUND: We previously demonstrated differential expression of nicotinic acetylcholine receptors (nAChRs) in the facial nerve-innervated orbicularis oris and somatic nerve-innervated gastrocnemius, which contribute to different sensitivities to muscle relaxants. Furthermore, the orbicularis oris exhibits less sensitivity to muscle relaxants after facial nerve injury, which is also related to upregulation of nAChRs. Here, we explored the regulatory mechanism for the different expression patterns. Because the agrin/Lrp4/MuSK/rapsyn and neuregulin1/ErbB signaling pathways are indispensable for maintaining the expression of nAChRs, we examined the activity of these two signaling pathways in gastrocnemius and orbicularis oris innervated by normal or injured facial nerves. MATERIALS AND METHODS: A quantitative analysis of these two signaling pathways was realized by immunofluorescence, and immunoprecipitation was applied to detect the level of phosphorylated MuSK in the gastrocnemius and orbicularis oris innervated by normal or injured facial nerves in adult rats. RESULTS: ErbB and the phosphorylated MuSK were expressed more in orbicularis oris than in gastrocnemius (P < 0.05). No significant difference was found in the expression of agrin/Lrp4/MuSK/rapsyn. After facial nerve injury, the level of agrin and the percentage of phosphorylated MuSK decreased significantly, although the expression levels of MuSK, rapsyn, and neuregulin1/ErbB were highly upregulated. CONCLUSIONS: Differential expression of the neuregulin1/ErbB signaling pathway may account for the different expression patterns of nAChRs at the neuromuscular junctions of the orbicularis oris and gastrocnemius. Overexpression of MuSK and rapsyn may contribute to upregulation of nAChRs after facial nerve injury.

Genioglossal response to mechanical vibrations of the mandible and the submandibular muscles.

The extrinsic tongue muscles are activated in coordination with pharyngeal muscles to dilate the airways as needed during breathing. The genioglossus (GG) activity is known to be modulated by several reflexes evoked via the mechanoreceptors of the upper airways. The primary objective of this paper was to investigate the effectiveness of activating these reflex pathways using mechanical stimulation of the mandible or the submandibular muscles. In eight healthy subjects, 3-s long, 5-mm vertical mechanical vibrations were delivered at 8 and 12 Hz to the lower jaw in a seated position, while the GG EMG was recorded using a custom-made sublingual electrode, along with the activity of the masseter (MS) and mylohyoid (MH). All three muscle activities were significantly higher during stimulation compared with the baseline (P < 0.02), and the increase was larger at 12 Hz versus 8 Hz (P < 0.02). All three muscle responses had components that synchronized with the mechanical stimuli, but those of MS were much more strongly phase-locked to the vibrational cycle. In 10 healthy subjects, we also applied mechanical vibrations to the submandibular muscles at three different stimulation intensities, while subjects were lying in a supine position. The GG activity increased significantly above the baseline (P = 0.026) in 9 out of 10 subjects, and the elevated activity persisted after termination of the stimulus for a few seconds. The results demonstrate that GG muscle responses can be evoked with mechanical vibrations applied to the lower jaw or the submandibular muscles in healthy subjects during wakefulness. NEW & NOTEWORTHY The evoked responses observed in the genioglossus (GG) activity during mechanical vibrations of the lower jaw or the submandibular muscles may lead to therapeutic applications for improving the patency of airways during sleep. The presence of these GG reflexes may also explain a mechanism by which the vibrations produced during snoring can help the airways stay open in individuals who may otherwise have obstructed airways in sleep.

Searching for proprioceptors in human facial muscles.

The human craniofacial muscles innervated by the facial nerve typically lack muscle spindles. However these muscles have proprioception that participates in the coordination of facial movements. A functional substitution of facial proprioceptors by cutaneous mechanoreceptors has been proposed but at present this alternative has not been demonstrated. Here we have investigated whether other kinds of sensory structures are present in two human facial muscles (zygomatic major and buccal). Human checks were removed from Spanish cadavers, and processed for immunohistochemical detection of nerve fibers (neurofilament proteins and S100 protein) and two putative mechanoproteins (acid-sensing ion channel 2 and transient receptor potential vanilloid 4) associated with mechanosensing. Nerves of different calibers were found in the connective septa and within the muscle itself. In all the muscles analysed, capsular corpuscle-like structures resembling elongated or round Ruffini-like corpuscles were observed. Moreover the axon profiles within these structures displayed immunoreactivity for both putative mechanoproteins. The present results demonstrate the presence of sensory structures in facial muscles that can substitute for typical muscle spindles as the source of facial proprioception.

Comparison of trophic factors' expression between paralyzed and recovering muscles after facial nerve injury. A quantitative analysis in time course.

After peripheral nerve injury, recovery of motor performance negatively correlates with the poly-innervation of neuromuscular junctions (NMJ) due to excessive sprouting of the terminal Schwann cells. Denervated muscles produce short-range diffusible sprouting stimuli, of which some are neurotrophic factors. Based on recent data that vibrissal whisking is restored perfectly during facial nerve regeneration in blind rats from the Sprague Dawley (SD)/RCS strain, we compared the expression of brain derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF2), insulin growth factors 1 and 2 (IGF1, IGF2) and nerve growth factor (NGF) between SD/RCS and SD-rats with normal vision but poor recovery of whisking function after facial nerve injury. To establish which trophic factors might be responsible for proper NMJ-reinnervation, the transected facial nerve was surgically repaired (facial-facial anastomosis, FFA) for subsequent analysis of mRNA and proteins expressed in the levator labii superioris muscle. A complicated time course of expression included (1) a late rise in BDNF protein that followed earlier elevated gene expression, (2) an early increase in FGF2 and IGF2 protein after 2 days with sustained gene expression, (3) reduced IGF1 protein at 28 days coincident with decline of raised mRNA levels to baseline, and (4) reduced NGF protein between 2 and 14 days with maintained gene expression found in blind rats but not the rats with normal vision. These findings suggest that recovery of motor function after peripheral nerve injury is due, at least in part, to a complex regulation of lesion-associated neurotrophic factors and cytokines in denervated muscles. The increase of FGF-2 protein and concomittant decrease of NGF (with no significant changes in BDNF or IGF levels) during the first week following FFA in SD/RCS blind rats possibly prevents the distal branching of regenerating axons resulting in reduced poly-innervation of motor endplates.

Dynamic change of myogenin in denervated rat mimetic muscle.

We previously reported double innervation of rat mimetic muscles with labeling of facial nuclei. However, whether denervated mimetic muscles are affected after such nerve repair is not known. Rats were divided into five groups: Group A, controls; Group B, complete facial palsy; Group C, complete facial palsy with repair using end-to-end neurorrhaphy; Group D, incomplete facial palsy; and Group E, incomplete facial palsy with repair using end-to-side neurorrhaphy. Preoperatively and postoperatively, facial palsy and myogenin (Myog) expression in mimetic muscles were evaluated. Expression peaked on day 7 in Group B but was lower in Groups C and D. Expression in Groups D and E was comparable on day 28, and each model's score showed characteristic changes. Myog expression in facial mimetic muscles increases with denervation and decreases with nerve repair. Determining Myog expression levels in mimetic muscles just after nerve repair may help surgeons predict postoperative prognosis in facial palsy.

Regional diffusion of botulinum toxin in facial muscles: a randomised double-blind study and a consideration for clinical studies with split-face design.

Despite the extensive use of botulinum toxin A (BoNTA) in medical and cosmetic treatments, the potential spreading of BoNTA to surrounding tissues remains unknown. A patient with hemifacial paralysis upon blepharospasm treatment with low dose of BoNTA, prompted us to investigate the spreading effect. A randomised, double-blind study was conducted in which 5 healthy women (33-52 years) were treated with different doses of onabotulinum toxin unilaterally in the corrugator muscle. Parameters of efficacy and diffusion (CMAP; EMG and jitter analysis) in both glabellar and frontalis muscles were assessed at baseline, 2 and 4 weeks following BoNTA injection. CMAP of the treated glabellar muscles was reduced to approximately 40% in all dose groups. Additionally, contralateral CMAP reduction was observed in 3 of 5 subjects. These data confirm regional diffusion of BoNTA in facial muscle application, which raises question on the reliability of split-face models in BoNTA studies.

Variety and complexity of fluorine-18-labelled fluoro-2-deoxy-D-glucose accumulations in the oral cavity of patients with oral cancers.

OBJECTIVES: To elucidate the points that require attention when interpreting fluorine-18-labelled fluoro-2-deoxy-d-glucose ((18)F-FDG)/positron emission tomography (PET) images by demonstration of (18)F-FDG accumulation in various areas of the oral cavity other than primary lesions in patients with oral cancers. METHODS: (18)F-FDG accumulations with a maximal standardized uptake value of over 2.5 in various areas of the oral cavity other than primary lesions were identified in 82 patients with oral cancers. RESULTS: (18)F-FDG/PET-positive areas, excluding primary tumours, included the front intrinsic muscles of the tongue (89.0%), upper and lower marginal parts of the orbicularis oris muscle (64.6%), sublingual glands, palatine tonsil, pharyngeal tonsil, and lingual tonsil. In addition, some areas in the jaws also showed accumulation. CONCLUSIONS: In patients with oral cancers, areas of (18)F-FDG accumulation in the oral cavity should be precisely identified and appropriately diagnosed, because accumulations can be seen in areas other than the primary tumour.

Small heat shock protein HSPB1 regulates growth of embryonic zebrafish craniofacial muscles.

The small heat shock protein HspB1 (Hsp27) is abundantly expressed in embryonic muscle tissues of a wide variety of vertebrate species. However, the functional significance of this expression pattern is not well established. In the present study, we observed specific, high level expression of HspB1 protein and an HspB1 gene reporter in developing craniofacial muscles of the zebrafish, Danio rerio, and examined the consequences of reducing HspB1 expression to the development and growth of these muscles. Quantitative morphometric analyses revealed a reduction in the cross-sectional area of myofibers in embryos expressing reduced HspB1 levels by as much as 47% compared to controls. In contrast, we detected no differences in the number of myofibrils or associated nuclei, nor the number, size or development of chondrocytes in surrounding tissues. We also did not detect changes to the overall organization of sarcomeres or myofibrils in embryos expressing reduced levels of HspB1. Together our results reveal a critical role for HspB1 in the growth of myofibrils and provide new insight into the mechanism underlying its developmental function.

Myogenin expression in facial muscle following damage to the facial nerve.

CONCLUSION: Gene analysis of facial muscle may be a promising way to detect denervation of facial muscle, helping to determine the prognosis of a facial palsy early in its progression. OBJECTIVES: In the treatment of intratemporal facial palsy, early diagnosis of neural damage is important in deciding about therapeutic modalities. In this study, we investigated the relationship between the severity of facial palsy and the level of myogenin expressed in the facial muscle. METHODS: The animals were divided into two groups, depending on whether the facial nerve was resected or compressed. Expression of myogenin mRNA was examined using real-time PCR and in situ hybridization of the facial muscle following the nerve damage. RESULTS: Increased expression of myogenin was observed in the nerve resection group, while no such increase was seen in the nerve compression group. In situ hybridization indicated that myogenin was expressed exclusively in satellite cells around the denervated muscle fibers.

Targeted gene transfer into rat facial muscles by nanosecond pulsed laser-induced stress waves.

We investigate the feasibility of using nanosecond pulsed laser-induced stress waves (LISWs) for gene transfer into rat facial muscles. LISWs are generated by irradiating a black natural rubber disk placed on the target tissue with nanosecond pulsed laser light from the second harmonics (532 nm) of a Q-switched Nd:YAG laser, which is widely used in head and neck surgery and proven to be safe. After injection of plasmid deoxyribose nucleic acid (DNA) coding for Lac Z into rat facial muscles, pulsed laser is used to irradiate the laser target on the skin surface without incision or exposure of muscles. Lac Z expression is detected by X-gal staining of excised rat facial skin and muscles. Strong Lac Z expression is observed seven days after gene transfer, and sustained for up to 14 days. Gene transfer is achieved in facial muscles several millimeters deep from the surface. Gene expression is localized to the tissue exposed to LISWs. No tissue damage from LISWs is observed. LISW is a promising nonviral target gene transfer method because of its high spatial controllability, easy applicability, and minimal invasiveness. Gene transfer using LISW to produce therapeutic proteins such as growth factors could be used to treat nerve injury and paralysis.

Conditioned cues for smoking elicit preparatory responses in healthy smokers.

RATIONALE: Smoking cues are theorized to be conditioned stimuli (CSs) formed by repeated pairing with drug. Smoking paraphernalia can elicit subjective and physiological responses in smokers, indicative of positive affect and motivation to consume. Although these responses are probably the result of conditioning, direct evidence from human conditioning studies with physiological measures of motivational valence is rare. OBJECTIVE: The present study investigated the motivational properties of experimentally conditioned cues for smoking. METHODS: Thirty-nine smokers completed a differential conditioning protocol. Abstract pictures were used as CSs and single puffs on a cigarette as unconditioned stimulus (US). Skin conductance responses and facial electromyography of the zygomatic, corrugator, and orbicularis oris muscles were measured during conditioning. RESULTS: The conditioned cue for smoking (CS+) elicited stronger skin conductance responses and more activity of the zygomatic and orbicularis oris muscles than the CS-. CONCLUSIONS: These results support the notion that through pairing with smoking, neutral stimuli acquire the ability to elicit preparatory physiological responses, which are assumed to play an important role in the maintenance of addiction and relapse in the natural environment.

Acceleromyographic monitoring of neuromuscular block over the orbicularis oris muscle in anesthetized patients receiving vecuronium.

STUDY OBJECTIVE: To evaluate the level of neuromuscular block acceleromyographically over the orbicularis oris muscle. DESIGN: Prospective, randomized, controlled study. SETTING: Operating room of a university-affiliated hospital. PATIENTS: 36 adult, ASA physical status I and II women scheduled for mastectomy with air-oxygen-isoflurane-fentanyl anesthesia. INTERVENTIONS: Patients were randomized to two groups. In the orbicularis oris group (n=18), the facial nerve was stimulated and movement of the orbicularis oris muscle was measured acceleromyographically. In the control group (n=18), adduction of the thumb was quantified mechanically. MEASUREMENTS: Onset and recovery of neuromuscular block caused by vecuronium 0.1 mg/kg were compared between the groups. MAIN RESULTS: Time to onset of neuromuscular block in the orbicularis oris group was significantly shorter than in the control group (176 + or - 52 vs. 220 + or - 34 sec, mean + or - SD; P = 0.004). Times to return of the first, second, third, or fourth (T1, T2, T3, or T4) response of train-of four (TOF), and recovery of T1/control were comparable between the groups. Train-of-four ratio (T4/T1) in the orbicularis oris group was significantly higher than in the control group 50 to 120 minutes after vecuronium administration (P < 0.05). CONCLUSION: Depth of neuromuscular block can be assessed acceleromyographically over the orbicularis oris muscle. Onset of neuromuscular block is quicker and recovery of TOF ratio is faster over the orbicularis oris muscle than at the thumb in patients receiving vecuronium.

The transcription factor Six1a plays an essential role in the craniofacial myogenesis of zebrafish.

Transcription factor Six1a plays important roles in morphogenesis, organogenesis, and cell differentiation. However, the role of Six1a during zebrafish cranial muscle development is still unclear. Here, we demonstrated that Six1a was required for sternohyoideus, medial rectus, inferior rectus, and all pharyngeal arch muscle development. Although Six1a was also necessary for myod and myogenin expression in head muscles, it did not affect myf5 expression in cranial muscles that originate from head mesoderm. Overexpression of myod enabled embryos to rescue all the defects in cranial muscles induced by injection of six1a-morpholino (MO), suggesting that myod is directly downstream of six1a in controlling craniofacial myogenesis. However, overexpression of six1a was unable to rescue arch muscle defects in the tbx1- and myf5-morphants, suggesting that six1a is only involved in myogenic maintenance, not its initiation, during arch muscle myogenesis. Although the craniofacial muscle defects caused by pax3-MO phenocopied those induced by six1a-MO, injection of six1a, myod or myf5 mRNA did not rescue the cranial muscle defects in pax3 morphants, suggesting that six1a and pax3 do not function in the same regulatory network. Therefore, we proposed four putative regulatory pathways to understand how six1a distinctly interacts with either myf5 or myod during zebrafish craniofacial muscle development.

Increased secretion of salivary glands produced by facial vibrotactile stimulation.

Patients with low-back pain can be evaluated immediately by means of an electrical tool that produces bony vibration to the lumbar spinal processes (Yrjama M, Vanharanta H. Bony vibrotactile stimulation: A new, non-invasive method for examining intradiscal pain. European Spine Journal 1994;3:233-235). In the rehabilitation of masticatory disturbance and dysphagia, an electric toothbrush is commonly used as an oral motor exercise tool for the facilitation of blood flow and metabolism in the orofacial region in Japanese hospitals. However, subjects receiving vibration in the facial regions reported increased salivary secretion. We attempted to develop an oral motor exercise apparatus modified by a headphone headset that was fixed and could be used for extended periods. The vibration apparatus of the heating conductor is protected by the polyethyle methacrylate (dental mucosa protective material), and electric motors for vibration control of the PWM circuit. We examined the amount of salivation during vibration stimuli on the bilateral masseter muscle belly, using a cotton roll positioned at the opening of the secretory duct for 3 min. Although the quantity of salivation in each subject showed various and large fluctuations in the right and left sides of the parotid and submandibular and sublingual glands, one or more of the salivary glands were effectively stimulated by 89 Hz vibration. The reported apparatus will be useful as an additional method in orofacial rehabilitation.

Fiber type and myosin heavy chain compositions of adult pretarsal orbicularis oculi muscle.

Pretarsal orbicularis oculi muscle (POOM) is an important structure of eyelid movement in human. The aim of this study was to investigate fiber histomorphology and myosin heavy chain (MyHC) isoform composition of adult POOM, and to clarify their age-related changes. Eyelid specimens from 58 subjects (age range, 21 to 91 years) were collected during upper blepharoplasty procedures. Serial cross sections of POOM were ATPase-stained and examined under miscroscope. Quantitative measures of muscle fiber size and fiber type distribution were obtained in 35 subjects with adequate fiber cross sections. Relative MyHC isoform contents of POOM were retrieved by gel electrophoresis in all 58 subjects. Examination of the histochemical staining revealed an abundance of type II fiber ( >85%) in human POOM, with more type IIX than IIA fibers. Decreased mean area of all fibers and type IIA fibers were noted in the old group when compared to the young. As for MyHC analysis, the relative content of MyHC isoforms exhibited an order of IIX > IIA > I, and the relative MyHC IIA content showed a negative correlation with age. Comparing with previous studies of limb or masticatory muscles, adult POOM exhibits a unique fiber and MyHC composition, as well as a different aging pattern.