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Innervation of the orbicularis oris muscle by the hypoglossal nucleus in rabbit
EXPERIMENTAL
NEUROLOGY
67, 247-250
(1980)
RESEARCH Innervation
of the Orbicularis Oris Muscle Hypoglossal Nucleus in Rabbit
IMAHARU Department
NOTE
NAKANO
AND TETSUO
FURUKAWA
of Neurology> Institute of Brain Research. Fuculty University of Tokyo, Tokyo, Japan Received
May
by the
of Medicine,
23, 1979
Unilateral orbicuhuis oris muscle of five albino rabbits was injected with horseradish peroxidase (HRP), which appeared in a number of cells of the ipsilateral facial nucleus. No granule of HRP was detected in cells of the hypoglossal nucleus. This result is contrary to the time-honored hypothesis that some cells of the hypoglossal nucleus innervate the orbicularis oris muscle.
It is well documented that some fibers from the hypoglossal nucleus take an ascending intramedullary path, join the facial nerve, and innervate the orbicularis oris muscle (3, 5). However, there appears to be no reliable experimental study on this matter. The following experiment was carried out to ascertain whether or not such an innervation is really present. Five young albino rabbits (about 500 g) were used for this experiment. About 200 ~15% solution of horseradish peroxidase (HRP, Sigma Type VI) in distilled water was injected into the left side of the orbicularis oris muscle. The next procedure followed the method of Nauta et al. (6). After 48-h survival, the animals were anesthetized intraperitoneally with pentobarbital(30 to 40 mg/500 g body wt) and were perfused by a massive intracardiac injection of 0.9% saline, followed by 1% paraformaldehyde-1.5% glutaraldehyde in 0.08 M phosphate buffer (PH 7.4). The brain stem was removed immediately after fixation and was fixed again 3 h with the same fixative at 4°C. It was stored at 4°C in the 0.2 M phosphate buffer (PH 7.4) Abbreviations:
HRP-horseradish
peroxidase, ALS-amyotrophic
lateral sclerosis
247 0014-4886/80/010247-04$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved
248
NAKANO
AND
FURUKAWA
containing 20% (w/v) sucrose. Next day, frozen transverse sections (100 pm) were made serially from the block. The procedure for demonstrating the presence of HRP is essentially that of Graham and Karnovsky (2). In the dark, the sections were preincubated 60 min at room temperature in 0.05% diaminobenzidine. Then, H202 was added to the solution to a final concentration of 0.01%. The sections were incubated 10 min at room temperature with stirring several times. They were washed with distilled water and were mounted on slides with glycerin-zeratin (1: 1). Most slides were left unstained. The others were counterstained with cresyl echt violet. It was confirmed that complete serial sections of hypoglossal nucleus were included in this block. The facial nucleus had been divided into two blocks in each case. The facial nucleus of the rabbit contains four subgroups (7). Figure 1 shows that HRP was detected mainly in the lateral subgroup of the ipsilateral facial nucleus. Ten to forty neurons were found to have taken up HRP per one section and 100 to 200 neurons per one animal, whereas HRP never appeared in the neurons of the hypoglossal nucleus nor in the small cell groups around this nucleus. In the 19th century, there was the hypothesis that some fibers from the hypoglossal nucleus ascend in the brain stem to join the facial nerve and supply the orbicularis oris muscle. There was a difference of opinion concerning this point among investigators. Oppenheim was against this and believed that such fibers, if present, were anomalous, whereas Tooth and Stewart supposed this theory (4). Grugia and Matteucei suggested the presence of anastomoses between facial and hypoglossal nerves, reporting a case in which both tongue and lips were paralyzed with a reaction of degeneration (1). In amyotrophic lateral sclerosis (ALS), the orbicularis oris is more apt to be involved than any other facial muscle and pathological examination often reveals considerable changes of the facial nerve in spite of an essentially intact facial nucleus. To explain these mysterious phenomena, the hypothesis was proposed that a group of small cells behind the hypoglossal nucleus supply their fibers to the orbicularis oris through the facial nerve. In ALS, the group of small cells as well as the hypoglossal nucleus are severely damaged and the lesion of the former results in paralysis of the orbicularis oris muscle. This explicitly explains the findings in ALS, but has not been proved. Bing stated that the lesion of the hypoglossal nucleus produced not only lingual paralysis but also ipsilateral paresis of the orbicularis oris. He drew a schema in which some fibers coming from the hypoglossal nucleus take an ascending intramedullary path to join the ipsilateral facial nucleus (3). Monrad-Krohn and Refsum (5) also mentioned the possibility of innervation of the orbicularis oris muscle by the hypoglossal nerve.
ORBICULARIS
ORIS
MUSCLE
INNERVATION
249
250
NAKANO
AND
FURUKAWA
Our experimentation revealed findings which did not support this hypothesis. Although a number of HRP-marked neurons were detected in the ipsilateral facial nucleus, neurons of the hypoglossal nucleus or the small cells around the nucleus showed no sign of HRP granules. This result suggests that the orbicularis oris of rabbits receives no fibers from the hypoglossal nucleus or that the five rabbits used in this study happened to have no such fibers because of variability of these fibers. Of course, this study on rabbits cannot be directly applied to man. But it is doubtful whether such an anastomosis really exists. REFERENCES 1. GOWERS, W. R. 1970. Diseases of the Brain: A Manual of the Nervous System, Vol. 2, 2nd. ed., pp. 49 and 570. Hafner, Darien. 2. GRAHAM, R. C., AND M. J. KARNOVSKY. 1966. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14: 291-302. 3. HUERTAS, J., AND W. HAYMAKER. 1959. Localization of lesions involving the facial nerve. Page 223 in W. HAYMAKER. Ed., Bing’s Local Diagnosis in Neurological Diseases, 15th ed. Mosby, Saint Louis. 4. MOLL, A. M. 1913. A case of hypoglossal nuclei paralysis. J. Nerv. Ment. Dis. 40: 179-181. 5. MONRAD-KROHN, G. H., AND S. REFSUM. 1964. Cranial Nerves: The Clinical Examination of the Nervous System, 12th ed. Lewis, London. 6. NAUTA, H. J. W., M. B. PRITZ, AND R. J. LASEK. 1974. Afferents to the rat caudoputamen studied with horseradish peroxidase: an evaluation of a retrograde neuroanatomical research method. Brain Res. 67: 219-238. 7. WINKLER, C., AND A. POTTER. 1911. An Anatomical Guide to Experimental Researches of the Rabbit’s Brain, Plate 30. W. Versluys, Amsterdam.
NEUROLOGY
67, 247-250
(1980)
RESEARCH Innervation
of the Orbicularis Oris Muscle Hypoglossal Nucleus in Rabbit
IMAHARU Department
NOTE
NAKANO
AND TETSUO
FURUKAWA
of Neurology> Institute of Brain Research. Fuculty University of Tokyo, Tokyo, Japan Received
May
by the
of Medicine,
23, 1979
Unilateral orbicuhuis oris muscle of five albino rabbits was injected with horseradish peroxidase (HRP), which appeared in a number of cells of the ipsilateral facial nucleus. No granule of HRP was detected in cells of the hypoglossal nucleus. This result is contrary to the time-honored hypothesis that some cells of the hypoglossal nucleus innervate the orbicularis oris muscle.
It is well documented that some fibers from the hypoglossal nucleus take an ascending intramedullary path, join the facial nerve, and innervate the orbicularis oris muscle (3, 5). However, there appears to be no reliable experimental study on this matter. The following experiment was carried out to ascertain whether or not such an innervation is really present. Five young albino rabbits (about 500 g) were used for this experiment. About 200 ~15% solution of horseradish peroxidase (HRP, Sigma Type VI) in distilled water was injected into the left side of the orbicularis oris muscle. The next procedure followed the method of Nauta et al. (6). After 48-h survival, the animals were anesthetized intraperitoneally with pentobarbital(30 to 40 mg/500 g body wt) and were perfused by a massive intracardiac injection of 0.9% saline, followed by 1% paraformaldehyde-1.5% glutaraldehyde in 0.08 M phosphate buffer (PH 7.4). The brain stem was removed immediately after fixation and was fixed again 3 h with the same fixative at 4°C. It was stored at 4°C in the 0.2 M phosphate buffer (PH 7.4) Abbreviations:
HRP-horseradish
peroxidase, ALS-amyotrophic
lateral sclerosis
247 0014-4886/80/010247-04$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved
248
NAKANO
AND
FURUKAWA
containing 20% (w/v) sucrose. Next day, frozen transverse sections (100 pm) were made serially from the block. The procedure for demonstrating the presence of HRP is essentially that of Graham and Karnovsky (2). In the dark, the sections were preincubated 60 min at room temperature in 0.05% diaminobenzidine. Then, H202 was added to the solution to a final concentration of 0.01%. The sections were incubated 10 min at room temperature with stirring several times. They were washed with distilled water and were mounted on slides with glycerin-zeratin (1: 1). Most slides were left unstained. The others were counterstained with cresyl echt violet. It was confirmed that complete serial sections of hypoglossal nucleus were included in this block. The facial nucleus had been divided into two blocks in each case. The facial nucleus of the rabbit contains four subgroups (7). Figure 1 shows that HRP was detected mainly in the lateral subgroup of the ipsilateral facial nucleus. Ten to forty neurons were found to have taken up HRP per one section and 100 to 200 neurons per one animal, whereas HRP never appeared in the neurons of the hypoglossal nucleus nor in the small cell groups around this nucleus. In the 19th century, there was the hypothesis that some fibers from the hypoglossal nucleus ascend in the brain stem to join the facial nerve and supply the orbicularis oris muscle. There was a difference of opinion concerning this point among investigators. Oppenheim was against this and believed that such fibers, if present, were anomalous, whereas Tooth and Stewart supposed this theory (4). Grugia and Matteucei suggested the presence of anastomoses between facial and hypoglossal nerves, reporting a case in which both tongue and lips were paralyzed with a reaction of degeneration (1). In amyotrophic lateral sclerosis (ALS), the orbicularis oris is more apt to be involved than any other facial muscle and pathological examination often reveals considerable changes of the facial nerve in spite of an essentially intact facial nucleus. To explain these mysterious phenomena, the hypothesis was proposed that a group of small cells behind the hypoglossal nucleus supply their fibers to the orbicularis oris through the facial nerve. In ALS, the group of small cells as well as the hypoglossal nucleus are severely damaged and the lesion of the former results in paralysis of the orbicularis oris muscle. This explicitly explains the findings in ALS, but has not been proved. Bing stated that the lesion of the hypoglossal nucleus produced not only lingual paralysis but also ipsilateral paresis of the orbicularis oris. He drew a schema in which some fibers coming from the hypoglossal nucleus take an ascending intramedullary path to join the ipsilateral facial nucleus (3). Monrad-Krohn and Refsum (5) also mentioned the possibility of innervation of the orbicularis oris muscle by the hypoglossal nerve.
ORBICULARIS
ORIS
MUSCLE
INNERVATION
249
250
NAKANO
AND
FURUKAWA
Our experimentation revealed findings which did not support this hypothesis. Although a number of HRP-marked neurons were detected in the ipsilateral facial nucleus, neurons of the hypoglossal nucleus or the small cells around the nucleus showed no sign of HRP granules. This result suggests that the orbicularis oris of rabbits receives no fibers from the hypoglossal nucleus or that the five rabbits used in this study happened to have no such fibers because of variability of these fibers. Of course, this study on rabbits cannot be directly applied to man. But it is doubtful whether such an anastomosis really exists. REFERENCES 1. GOWERS, W. R. 1970. Diseases of the Brain: A Manual of the Nervous System, Vol. 2, 2nd. ed., pp. 49 and 570. Hafner, Darien. 2. GRAHAM, R. C., AND M. J. KARNOVSKY. 1966. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14: 291-302. 3. HUERTAS, J., AND W. HAYMAKER. 1959. Localization of lesions involving the facial nerve. Page 223 in W. HAYMAKER. Ed., Bing’s Local Diagnosis in Neurological Diseases, 15th ed. Mosby, Saint Louis. 4. MOLL, A. M. 1913. A case of hypoglossal nuclei paralysis. J. Nerv. Ment. Dis. 40: 179-181. 5. MONRAD-KROHN, G. H., AND S. REFSUM. 1964. Cranial Nerves: The Clinical Examination of the Nervous System, 12th ed. Lewis, London. 6. NAUTA, H. J. W., M. B. PRITZ, AND R. J. LASEK. 1974. Afferents to the rat caudoputamen studied with horseradish peroxidase: an evaluation of a retrograde neuroanatomical research method. Brain Res. 67: 219-238. 7. WINKLER, C., AND A. POTTER. 1911. An Anatomical Guide to Experimental Researches of the Rabbit’s Brain, Plate 30. W. Versluys, Amsterdam.