Journal of the Anatomical Society of India

Department of Anatomy, Stanley Medical College, Chennai. INDIA

Abstract:

The serous, mucous, and mixed glands and parasympathetic ganglion cells are present in animal and human tongue. Anterior two thirds of the tongue of the monkey (Macaca radita) was removed and fixed in Zenker's fluid. Ten-micron thick serial transversesections were stained by Heidenhain's azocarmine aniline blue and Harris' haematoxylin methods. In between the muscle fibres, close tothe inferior surface of the tongue, were lingual glands and parasympathetic ganglion cells. Stimulation of the hypoglossal nerve, even afterlingual nerve axotomy, showed secretion from the lingual glands on the same side as stimulation. Presence of the lingual glands andparasympathetic ganglia and secretion from the lingual glands on stimulation of the hypoglossal nerve in the monkey (Macaca radiata) isdemonstrated and reported for the first time.

Key words: Dorsal motor nucleus, ganglion cells, hypoglossal nerve, lingual glands, monkey, tongue, vagus nerve

Introduction:

The lamina propria of the tongue extends between the lingual muscle fasciculi and contains the lingual glands of mucous, serous, and mixed types, small blood vessels, and nerves. The parasympathetic preganglionic neuron cell bodies are in the brain stem; the postganglionic neuron cell bodies are in the peripheral parasympathetic ganglia, which are associated with certain cranial nerves and innervate the viscera, glands, blood vessels, and smooth muscles. Small subsidiary ganglia occur near these ganglia (Baumann and Gajisin, 1975).

The existence of nerve ganglia, within the musculature of the mammalian tongue was known (Barker, 1899). The ganglia were found along deeply placed nerve bundles in the tongue of the cat, rabbit, rat, and hedgehog; some were located close to terminal branches of the hypoglossal nerve (Gerne and Garwan, 1952). Groups of nerve cells were found in the tongue musculature of cat, lamb, monkey, and man; larger ganglia were also found on the chordalingual; and glossopharyngeal nerves, close to the point of entry into the tongue (Cooper, 1953). Nerve cells were near the intramuscular nerve trunks in the human tongue; their processes were traced to the minor salivary glands in the musculature and to the arteries in the submucosa (El- Rakhawy and Bourne, 1961). Intramuscular nerve cells in several mammals resembled the autonomic nerve cells. (Wetzig, 1962, 63) Isolated nerve cells were in the post-sulcal region and were perhaps the postganglionic parasympathetic neurons, innervating glandular tissue and vascular smooth muscle (Chu, 1968). The ganglionic cells resembled those of the submandibular ganglion in man and dog and the ganglia were considered to be the distally displaced submandibular ganglion cells. The innervation of the lingual glands is from the chorda tympani branch of the facial nerve, snapping in the submandibular ganglion and distributing via the lingual nerve branches (Williams et al, 1995).

Whether the hypoglossal nerve contains parasympathetic nerve fibres is not clear. Presence of the lingual glands and paraympathetic ganglia has not been reported in the monkey (Macaca radiata). The purpose of this study was to find whether or not the lingual glands and parasympathetic ganglia were present in the tongue of the monkey (Macaca radiata) and if present, whether preganglionic parasympathetic fibres were in the hypoglossal nerve.

Materials and Methods:

Five adult monkeys (Macaca radiata) were used to demonstrate the intralingual glands and ganglia and another five monkeys for physiological experiments on the paraysympathetic component of the hypoglossal nerve.

Light micropscopy of the tongue.

The animals were anaesthetized under thiopentone sodium (30 mg/kg I.V) and perfused with normal saline. Anterior two thirds of the tongues were removed and fixed in Zenker's fluid (Zenker 1894). The tissues were processed for light microscopy. Ten-micron thick serial transverse paraffin sections were cut, mounted on slides, stained with Heidenhain's azocarmine aniline blue and Harris' haematoxylin methods, and viewed under microscope.

At the end of the physiological experiments, the anterior two thirds of the tongue was removed and processed for histological confirmation of secretions within the glands. Helly's fixative and PAS and azocarmine stains were used.

Physiological experiments:

Under thiopentone sodium anaesthesia (30 mg/kg I.V), the lingual nerve was exposed bilaterally in the neck below the mandible. Axotomy of the lingual nerve was done proximal to its communication with the hypoglossal nerve. A small bit of the nerve (0.5cm) was cut and removed. The wound was closed layer by layer. During the post-operative period, the animal was observed for thirst reaction by keeping known quantity of water by its side. The physiological experiments to test the secretory nature of the hypoglossal nerve were done 7 to 10 days after the lingual nerve axotomy.

The animal was anaesthetised by thiopentone sodium and tubocurarine (3mg/kg) was injected through the lingual artery to relax the tongue muscles. The tongue was wiped with cotton. The hypoglossal nerve was stimulated bilaterally for one minute with sqaure wave pulses of 6-volt amplitude and 2-millisecond pulse width. Any secretion that formed in the anterior two thirds of the tongue was collected on blotting papers, which had been previously weighed. The blotting papers were immediately weighed. Following this, the hypoglossal nerves were divided bilaterally and the distal cut end was stimulated as before. The secretion in the anterior two thirds of the tongue was collected on blotting papers, which was immediately weighed. In two animals, after the stimulation of the intact hypoglossal nerves, atropine (1mg/kg) was injected through a systemic vein. The hypoglossal nerves were again stimulated for one minute at an interval of one minute. The secretion was collected and weighed as before.

Observations:

Light microscopy of tongue:

The lingual glands were present. The glands were of mucous and serous types. In between the muscle fibres, close to the inferior surface of the tongue, collections of nerve cells were present in the lamina propria; groups, each of twenty five to thirty nerve cells, formed the ganglia; the ganglia were surrounded by capsules and were close to the lingual glands (Fig.1). The nerve cells were larger than the connective tissue stromal cells and were ovoid or polygonal and multipolar; the nucleus was spherical, vesicular, and eccentric in position; the scanty chromatin granules were distributed along the nuclear membrane; the nucleolus was prominent; each cell was surrounded by a number of satellite cells; the cytoplasm appeared granular; Nissle bodies were present; nerve fibres arose from these cells (Fig.2). Occasionally, the ganglion cells were present close to the blood vessels.

Physiological experiments:

The bilateral lingual nerve axotomy did not produce any change in the thirst reaction even after 7 to 10 days after the operation. There was no difference in the intake of water between the control and experimental animals.

When the intact hypoglossal nerve was stimulated on one side, the secretions poured along the margins as well as the under surface of the tongue on the stimulated side. The secretion was like sweat coming from the sweat pores. The quantity varied from 12 to 19 mgm on the left side and 20 to 22 mgm on the right side (Table 1).

Table 1: Weights of the secretions on stimulation of the hypoglossal nerve after bilateral lingual nerve axotomy.

When the hypoglossal nerves were divided on both sides and their distal cut ends were stimulated individually on either side, the secretions were less in quantity when compared with that of intact hypoglossal nerve stimulation

Subsequently, PAS and azocarmine positive secretory materials were histologically demonstrated in the acini and ducts of the lingual glands. The glands were filled with the secretions after the stimulation of the hypoglossal nerve (Fig. 3)

Effect of atropine

The stimulation of the intact hypoglossal nerve, one minute before the intravenous administration of the atropine resulted in copious secretion varying from 9 to 22 mg but in the same animals, stimulation of the hypoglossal nerve, one minute after the intravenous administration of the atropine, resulted in a negligible secretion, varying from 0.05 to 0.2 mg (Table 2).

Table 2: Weights of the secretions on stimulation of the hypoglossal nerve before and after administration of atropine.

Discussion:

Tongue:

The presence of the ganglia in the tongue was reported in the pig (Kane 1952), human (Gerne and Garvan 1952; Sakla 1964) Japanese dormouse (Kubota and Togava, 1966), rat (Chang, Wang and Lu, 1966, Chu, 1968), cat (Fitzgerald and Alexander, 1969), safed teeter (Purwar, 1975), goat (Qayyum and Beg, 1975), cyanamolgus monkey (Fitzgerald and Sachithanandan, 1978) and Indian Buffalo (Prakash and Rao, 1980) Ganglia and mixed, serous and mucous lingual glands were present in the tongues of the great ant eater (Kubota et al, 1962a), pangolin (Kubota et al, 1962b), marsupials (Kubota et. al, 1963), pigmy and common marmosets (Kubota and Kayama, 1964), Japanese pika alongside the nerve fibre bundles (Kubota, 1966) and Japanese dormouse on the nerve bundles in the muscles (Kubota and Togawa, 1966).

Physiological studies:

After the division of the lingual nerves, the only nerve in contact with the anterior two thirds of the tongue was the hypoglossal nerve. Any reflexes should necessarily pass through that nerve only. Contraction of muscle fibres causing secretion was prevented by tubocurarine. Any secretion that occurred in the anterior two thirds of the tongue, on stimulation of the hypoglossal nerve, could be only due to the connection of that nerve. When the distal cut end of the hypoglossal nerve was stimulated also, small amounts of acetylecholine, liberated at the nerve endings, could have caused the secretion. Histologically also, the secretion is shown in the gland. Administration of atropine, a parasympatholytic agent, blocked the secretion.

Preganglionic fibres:

In the posterior third of the human tongue, many solitary parasympathetic neurons supplied adenomotor fibres to von Ebner's gland and myomotor to smooth muscles of blood vessels (Chu, 1968). The ganglia were throughout the tongue musculature in the cat and the chorda-lingual nerve was the main source of the pregaglionic fibres (Fitzerald and Alexander, 1969). The ganglia functionally connected with the chorda tympani nerve. In the Syrian hamster, the intralingual ganglia were dispersed along the ramifications of the lingual nerve (Kuder and Szczurkowski, 1997). The intralingual ganglia were studied in the porcine, monkey and canine tongue (Yoshida and Toda, 1997). In the cat, the ganglia were innervated by fibres from the pterygopalatine ganglion (Ito and Oyagi, 1994). Parasympathetic postganglionic cells were in the lingual branch of the glossopharyngeal nerve (Lnoue and Kitada, 1991). The lingual ganglia consist of parasympathetic neurons (Chibuzo et. al, 1980).

The nerve cells on the hypoglossal nerve resemble the cells in the inferior ganglion of the vagus nerve and are always observed within the communicating rami between this ganglion and the hypoglossal nerve; possibly, these cells migrate from the inferior ganglion of the vagus to the hypoglossal nerve for proprioceptive innervation of the tongue (Wozniak and Young, 1968): Probably, the ganglion cells in the tongue migrate from the neural crest along the vagus nerve, its communications, and hypoglossal nerve to the tongue and relay in the lingual ganglion to supply the lingual glands.

In the present study, the presence of the lingual glands and intralingual parasympathetic ganglia show that the hypoglossal nerve contains preganglionic parasympathetic fibres and the fibres synapse with the intralingual ganglia to supply the intralingual glands and vascular smooth muscles. The presence of the lingual glands and parasympathetic ganglia and the neural pattern of the parasympathetic fibres in the hypoglossal nerve to distribute to the tongue in the monkey (Macaca radiata) is demonstrated and reported for the first time.

Acknowledgement:

The author thanks Dr. M. Muthu and Dr. Leela Rajendran for their help in this study.

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Fig. 1

Section of anterior two thirds of the tongue. Parasympathetic ganglion (arrow); GL - lingual glands; M - lingual muscle. Azocarmine aniline blue 20 X.

Fig 2

Parasympathetic ganglion cell. GC - ganglion cell; Satellite cell (arrow). Azocarmine aniline blue 800X.

Fig.3

Section of anterior two thirds of tongue after lingual nerve axotomy and stimulation of hypoglossal nerve. GL - lingual glands filled with secretion; M - muscle fibres. PAS 20 X.

Fig. 4

Section of lingual glands. Glands filled with secretory materials. PAS 80 X.