Quantitative studies of the greater petrosal nerve of the mouse with the electron microscope.код для вставкиСкачать
Quantitative Studies of the Greater Petrosal Nerve of the Mouse with the Electron Microscope ATSUMI SHIMOZAWA Department of A n a t o ~ n y ,School of Medicine, Chiba University, Chiba, J a p a n ABSTRACT Quantitative counts of the greater petrosal nerve fibers of the mouse with the electron microscope have shown that, on an average, 70.8% and 28.7% of the total nerve fibers (1,111) were unmyelinated and myelinated fibers respectively. Unexpected high incidence of unmyelinated fibers in the greater petrosal nerve may indicate that it contains a fair amount of sympathetic postganglionic fibers. The unmyelinated fibers in the nerve may well represent parasympathetic preganglionic fibers. The presence of unmyElinated fibers in the greater petrosal nerve of the cat has been noted by Foley and DuBois ('43) with the light micrcscope, a fact which seems to clarify the nature of the fibers constituting the greater petrcsal nerve. However, quantitative counts of the unmyelinated fibers have been difficult with the light microscope. An application of electron microscopy for the fiber count was made in this study so as to determine the exact number of unmyelinated fibers within the greater petrosal nerve. a n entire area of the greater petrosal nerve cut transversely. OBSERVATIONS I n transverse sections, the entire greater petrosal nerve showed almost elliptical form with major and minor diameters of 49-150 and 28--48 respectively (fig. 2, table 1). The nerve was extremely flattened near the portion of its intermediate flexion (fig. 1). Out of the eight nerves examined i n this study, two were composed of two nerve bundles, large and , MATERIALS AND METHODS Four female adult mice weighing 1822 gm were perfused through the heart with 2.5% glutaraldehyde in Millonig's phosphate buffer at pH 7.5 (Millonig, '62; Sabatini et al., '64). The greater petrosal nerve was then dissected out under the binocular microscope and a proximal part of the nerve less than 1.3 m m from the geniculate ganglion excised and postfixed in a 1% osmium tetroxide i n Millonig's phosphate buffer (fig. 1). After dehydration with ethanol, the specimen was embedded in Epon 812. Transverse sections of the nerve cut on a Porter-Blum microtome were mounted on a 100-mesh grid coated by collodion, stained with uranyl acetate and lead citrate (Reynolds, '63) and viewed in a Hitachi HS-8 electron microscope. Counts of myelinated and unmyelinated nerve fibers were made on a montage of electron micrographs covering ANAT. REC., 170: 303-308. N. c a n a l i s pterygoidei . carotis interna N. petrosus major Ggl. geniculi N. interrnedius Facial trunk Fig. 1 A dorsal view of the right greater petrosal nerve of the mouse. The striped part of the nerve was used as material in this study. Received Aug. 31, '70. Accepted Dec. 8, '70. 303 304 ATSUMI SHIMOZAWA Fig. 2 A montage of electron micrographs covering an entire area of the greater petrosal nerve of the mouse (no. 4, L ) cut transversely. QUANTITATIVE STUDIES OF N. PETROSUS MAJOR 305 Fig. 3 A higher magnification of tlte region outlined in figure 2. M, Myelinated nerve fibers; U, unmyelinated nerve fibers. small (no. l L , no. 3L). Within the nerve bundle enclosed by the perineural sheath, a blood capillary was found in two of the eight nerves (no. 2L, no. 4R), and some- times a few scattered ganglion cells were encountered at the locus near the geniculate ganglion. No communicating branches to or from the greater petrosal nerve were 306 ATSUMI SHIMOZAWA TABLE 1 A fiber atzalysis of the greater petrosat nerve of the mouse, as shown by electron microscopy Diameter of the nerve Animal no. Side Distance from ggl. genic. Minor Major Myelinated Unmyelinated Undetermined Total mm Right Left 0.4-1.0 0.5-1.0 35 38 105 110 243 239 720 784 3 2 966 1025 R L 0.5-1.0 0.0 40 35 49 82 215 247 725 680 5 6 945 933 R 0.0-0.2 0.2-0.4 55 39 95 99 335 293 1049 1037 5 6 1389 1336 1.1-1.3 0.0 28 48 150 107 497 487 621 677 8 7 1126 1171 319 (28.7) 787 (70.8) L R L 5 (0.5) 1111 TABLE 2 Minor and major diameters of myelinated fibers i n the greater petrosal nerve of the mouse - Animal no. 1 2 3 4 Side 1-2p 2-3p 3-4p 10 185 37 162 20 47 134 69 103 1 59 6 85 Minor Major Minor Major 67 6 21 1 148 173 217 59 36 9 141 45 1 Minor Major Minor Major 3 227 30 209 12 101 164 79 149 4 114 1 113 27 423 148 392 139 45 269 87 277 79 4 67 Minor Major Minor Major Minor Average (%) 4-5p 0-1p Minor Major Minor Major Major 2 4 29 4 245 55 17 (5.3 (76.8’1 . , (17.3 . 1 77 159 (0.3) (24.1) (49.9) noticed under the binocular microscope at the time of dissection. Intermingled within the greater petrosal nerve were the myelinated and unmyelinated fibers that measured in minor diameters 0.6-3.4 EL and 0.1-1.0 EL respectively. These fibers generally were round, oval or elliptical in form, but some myelinated fibers showed an irregular contour at the sectional level normal to their fiber axis (fig. 3 ) . Although the thickness __ 5-6p Total 243 13 29 6-7p 1 239 215 2 (0.6) 70 19 1 4 247 335 293 497 487 319 12 of individual myelin sheaths varied to a considerable extent, it was rare to find such a myelinated fiber surrounded by less than three myelin lamellae. Quantitative fiber counts of the eight greater petrosal nerves from four mice (table 1) showed that, on an average, each nerve contained a total of 1,111 fibers, of which only 319 (28.7% ) were myelinated whereas 787 (70.8% ) had no investment of the myelin sheath at all. In addition, QUANTITATIVE STUDIES OF N. PETROSUS MAJOR there were on the average five profiles undetermined as to whether myelinated or not. Most of them showed a collar of processes of Schwann cells around the axiscylinder indicating that they may possibly represent a transverse sectional level through the node of Ranvier. The myelinated fibers in the greater petrosal nerve ranged from 0.6-3.4 in minor diameters with the majority (76.8% ) being 1-2 p, while their major diameters varied from 0.8-6.3 with those measuring 2-3 p being most frequent (table 2). DISCUSSION It is widely accepted that the greater petrosal nerve contains not only secretory fibers to the lacrimal gland, but also parasympathetic and sensory fibers innervating the mucous membrane of the nasal cavity and palate, as well as some taste fibers (Beninghoff-Goerttler, '57; Gray, '58; Mitchell, '53; Morris, '53). With regard to the relation between form and nature of the nerve fiber, it has been believed that, at most, the autonomic preganglionic fibers are thinly myelinated and the postganglionic fibers are unmyelinated, whereas the sensory fibers are myelinated with quite a variable diameter (Bloom and Fawcett, '68). Therefore, it seems that the myelinated fibers, making up about 30% of the total fibers in the greater petrosal nerve of the mouse, represent the parasympathetic preganglionic fibers to the lacrimal gland, the sensory and parasympathetic preganglionic fibers innervating the nasal and palatine mucosa, and some taste fibers as well. In a previous study of the myelinated fibers in the greater petrosal nerve of the crab-eating monkey (Shimozawa, '68) with a fan-wise split preparation method (Fukuyama, '54), it was shown that the nerve contained thick and thin myelinated fibers in almost equal quantities. Because of the difference of the methods used, i t is difficult to compare the results in the present study for the mouse with those in the previous study. However, it seems that the proportion of thin myelinated fibers in the greater petrosal nerve of the mouse may be more than that in the crab-eating monkey. 307 On the nature of the unmyelinated fibers, representing about 70% of the total number of fibers in the greater petrosal nerve, the following three probabilities are considered; ( 1 ) the sympathetic postganglionic fibers, ( 2 ) the parasympathetic preganglionic fibers, ( 3 ) the parasympathetic post ganglionic fibers. Although sympathetic fibers in the greater petrosal nerve have not been reported, their presence in the facial trunk distal to the geniculate ganglion has been shown in the cat by Foley ('48). The greater petrosal nerve unites with the deep petrosal nerve which contains a large number of the sympathetic postganglionic fibers, so it is probable that some fibers corresponding with those of the deep petrosal nerve may come into the facial nerve somewhere and pass through the greater petrosal nerve. Foley and DuBois ('37) have shown the presence of unmyelinated parasympathetic preganglionic fibers in the vagus nerve of the cat. Subsequently, after a section of the root of the facial nerve of the cat, they demonstrated that about 62% of the fibers of the greater petrosal nerve are visceral motor fibers, 80-90% of which are myelinated. So it seems also probable that the unmyelinated fibers in the greater petrosal nerve of the mouse may in part represent the parasympathetic preganglionic fibers. Okinaka et al. ('50) have shown that in the dog vagus nerve the unmyelinated fibers increased distal to the superior and inferior ganglia; the authors regarded many of the unmyelinated fibers in the vagus nerve as postganglionic in nature. However, the presence of parasympathetic postganglionic fibers in the greater petrosal nerve may be eliminated based on the findings that the geniculate ganglion i s composed of unipolar cells (Rhinehart, '18). Multipolar cells have not been yet iound in the ganglion. Thus, i t is postulated that the greater petrosal nerve of mice contains a considerable amount of the sympathetic postganglionic fibers and that some of the parasympathetic preganglionic fibers in the greater petrosal nerve may well be unmyelinated. Further experiments must be done to test the above postulations. 308 ATSUMI SHIMOZAWA ACKNOWLEDGMENTS The author wishes to thank Dr. U. Fukuyama, Dr. T. Nagano and Dr. A. Yamauchi for their help and advice. LITERATURE CITED Benninghoff, A., and K. Goerttler 1957 Lehrbuch der Anatomie des Menschen. 5 Aufl., Urban & Schwarzenberg. Muunchen-BerlinWien, 111: 302-303. Bloom, W.,and D. W. Fawcett 1968 A Textbook of Histology, 9th ed. W. B. Saunders Co. Philadelphia-London-Toronto. pp. 327-333. Foley, J. O., and F. S. DuBois 1937 Quantitative studies of the vagus nerve in the cat. J. Comp. Neur., 67: 49-68. 1943 A n experimental study of the facial nerve. J. Comp. Neur., 79: 79-105. Foley, J. 0. 1948 The special visceral efferent zone of the seventh cranial nerve i n the canalis facialis. J. Conip. Neur., 88: 4 3 9 4 5 1 . Fukuyama, U. 1954 O n the constituents of the hypogastric nerve. Fukushima J. Med. Sci., I: 117-139. Johnston, T. B., D. V. Davis and F. Davis 1958 Gray’s Anatomy, 32nd ed. Longmans, Green & Co., London, New York, Toronto. p. 1,118. Millonig, G. 1962 Further observations on a phosphate buffer for osmium solutions in fixation. Proc. 5th Intern. Cong. Electron Microscopy, Philadelphia, Vol. 11, Academic Press, New York. p. 8. Mitchell, G. A. G. 1953 Anatomy of the Autonomic Nervous System. 1st ed. E & S Livingstone Ltd. Edinburgh and London, pp. 162-164. Okinaka, S. 1950 O n the morphology of the autonomic nerve fibers. Nippon Rinsho, 8: 12-21. Reynolds, E. 1963 The use of lead citrate at high pH as a n electron-opaque stain in electron microscopy. J. Cell Biol., 17: 208-212. Rhinehart, D. A. 1918 The nervus facialis of the Albino mouse. J. Comp. Neur., 30: 81-125. Sabatini, D. D.,F. Miller and R. J. Barrnett 1964 Aldehyde fixation for morphological and enzyme histochemical studies with the electron microscope. J. Histochem. and Cytochem., 12: 57-71. Schaeffer, J. P. 1953 Morris’ Human Anatomy. 11th ed. McGraw-Hill Co., New York. 1102-1 104. Shimozawa, A. 1968 Myelinated nerve fibers i n the facial nerve of the crab-eating monkey. Acta Anat. Nipponica, 43: 73-88.