Cord by Resin Cracking Method. Kenji OHTSUKI

Transcription

1 Arch. histol. jap., Vol. 34, No. 4 (1972) p Department of Anatomy (Prof. K. TANAKA), Tottori University School of Medicine, Yonago, Japan Scanning Electron Microscopic Studies on Rabbit's Spinal Cord by Resin Cracking Method Kenji OHTSUKI Received March 23, 1972 Summary. Small pieces cut from spinal cord of rabbit fixed dehydrated embedded in gelatin capsules filled with Cemedine The Cemedine was specimen was observed under scanning electron microscope. 1. In cytoplasm of anterior horn cell Nissl bodies observed like specks. The Nissl bodies denser than cytoplasm surrounding m contained numerous or. In cytoplasm corresponding to Golgi regions many vesicles cisternal membranous structures could be seen. On surface of nerve cell bodies small swollen terminations of axons, which seemed to be end-bulbs, observed. 2. The membranes consisting of myelin sheaths, which surrounded axon as a whirlpool, observed in three dimensions. The myelin sheath sometimes extended thinner processes which could be considered to be cytoplasmic processes of neuroglial cell. 3. The cilia covering ependymal cells of central canal fairly uniform in number (12-15) from cell to cell, arranged in a tuft for each cell. The surface was also rich in microvilli which formed various shapes. The usefulness of scanning electron microscope in revealing fine details of surface morphology of cells tissues has been already established. Recently, scanning electron microscopic studies of nervous tissue have been reported on chick spinal cord (BOYDE et al., 1968), cilia in brain (DALEN et al., 1971), isolated neurons (HAMBERGER et al., 1970), isolated nerve fibers (SPENCER LIEBERMAN, 1971). However, all of se studies have treated with surface morphology of cells tissues, re have been no studies performed on inner structure as far as is known. In present study on anterior horn cells, neuroglia cells myelinated nerve fibers of spinal cord as observed with scanning electron microscope, author attempted not only to reveal surface morphology of cells tissues but also ir interior structure, making use of TANAKA's resin cracking method. Materials Methods Adult rabbits weighing kg used. Under er anessia rabbits perfused via abdominal aorta with 300ml of warmed physiological saline n with 500ml of 2.5% glutaraldehyde buffered with phosphate (ph 2mm) dissected out. The specimens fixed in 5% glutaraldehyde buffered with phosphate (ph 7.4) for 2hrs at room temperature, subsequently postfixed in 405

2 406 K. OHTSUKI: 1% osmium drated tetroxide in graded buffered ethanol, with veronal-acetate immersed (ph in propylene 7.4) for oxide 3hrs at 4, for 15min. dehy- The resin cracking method (TANAKA, 1972) was n applied. In this method specimens embedded in No. 2 capsules filled with Cemedine 1500 (epoxy resin). The Cemedine in capsules was Cemedine, pieces hardened of capsules specimens. in a cryostat capsules by critical point drying dried by acetone drying surfaces of cracked examined under HSM-2 sufficient transferred drying, in a vacuum scanning electron The Cemedine to amyl carbon hardening hammer. (ANDERSON, 1951). After specimens a Hitachi method method. After a chisel oxide to remove specimens dried with put into propylene Subsequently at -30. cracked of broken from acetate, Some specimens evaporator gold n applied material to was microscope. Observations Nerve cells The anterior vation. The cell bodies horn cells of spinal cord chosen as subject of obser- motor cells of generally anterior horn pyramidal or polygonal round nucleus located in center of cell body. in of mass of Fig. 1. nucleus clearly nerve nerve demarcated cell A scanning bodies electron cell from small was nucleolus, extremely in shape. of karyoplasm swollen terminations showing anterior neurons, They had a large, The most conspicuous object which rest micrograph large of was an extremely (Fig. 1). axons, which horn of spinal dense On surface seemed cord. Two to be nerve cells, which have a large cell body round nucleus with a conspicuous nucleolus. a blood vessel ate seen. 1,300

3 Scanning Fig. 2. A scanning electron Electron micrograph showing of a nerve Fig. 3. Cytoplasm of an anterior horn cell. (M) Microscopy are cell of Spinal end-bulbs (N). Cord (E) which are attached 407 to surface 5,000 Nissl bodies (N), Golgi complex (G) mitochondria observed. 20,000

4 408 K. OHTSUKI: end-bulbs, could often be seen (Fig. 2). These mainly observed in specimens which considerably shrunk due to dehydration procedure (acetone drying method). In cytoplasm of anterior horn cells, large clumps observed, similar to those that visible with aid of light microscope. These clumps identified with what are generally termed Nissl bodies. The Nissl bodies denser in architecture than cytoplasm surrounding m y contained concerned with ribosomes. Also membranous structures observed in lines in Nissl bodies which thought to be rough-surfaced endoplasmic reticulum. On or h, in cytoplasm around Nissl bodies, especially close to nucleus, re existed many vesicles cisternal membranous structures. These thought to be Golgi complex. The mitochondria generally disposed in cytoplasm between Nissl bodies, cristae mitochondriales identified on ir cracked surface (Fig. 3). Myelinated nerve fibers The white matter of spinal cord consisted of myelinated nerve fibers supported by neuroglial cells which lay between myelinated nerve fibers. Under scanning electron microscope it was clearly seen that cylindric myelinated nerve fiber bundles ran up down cord. On cross section of nerve fibers, axon myelin sheath could be clearly differentiated Fig. 4. A scanning electron micrograph showing a cross section of myelinated nerve fibers. Myelin

5 Scanning Fig. 5. Electron Microscopy Surface of myelinated nerve fibers. of Spinal A branching Cord 409 process (P) some fibrils are seen. 4,600 Fig. 6. A neuroglial cell (G) in gray matter. myelinated nerve fibers It exists close to a nerve cell (N) has groups (F) around it. 8,500

6 410 K. OHTSUKI: under scanning electron microscope. The myelin sheath consisted of membranes which surrounded axon in a whirl generally looked wavy. The axon appeared like a sponge was surrounded by axolemma. Sometimes objects which supposed to be mitochondria could be seen in axon (Fig. 4). The fibrils which seemed to be fine collagen fibers could be seen on outerlayer of myelin sheaths. Occasionally myelin extended a thin branch which was assumed to be derived from cell body of a neuroglial cell (Fig. 5). Neuroglial cells Some neuroglial cells observed in gray matter. Frequently seen satellite cells in close association with nerve cells. The cell bodies round or oval in shape far smaller than those of nerve cells. They had many processes also often associated with groups of myelinated nerve fibers (Fig. 6). In white matter, cells with a large round nucleus comparatively rich cytoplasm occurred in rows between myelinated nerve fibers (Fig. 7). Ependymal cells The ependymal cells around central canal of spinal cord consisted of a sheet of epilial cells. Many cilia numerous microvilli covered free surface of ependymal cells re seemed to be no difference in number of cilia from cell to cell. Some cilia which built up a tuft on ependymal cell derived from center of cell surface. Such tufts resembled young rice plants Fig. 7. A neuroglia cell in white matter exists between myelinated nerve fibers (F) has a round

7 Scanning Electron Microscopy of Spinal Cord 411 They smooth surfaced terminated in a rounded tip. The microvilli far shorter than cilia in length formed various shapes; ir tip was exped like a drumstick. The microvilli thickly grown around center of cell surface in same manner as cilia, but scarcely seen at periphery of cell surface (Fig. 9). Discussion A distinguishing feature of motor nerve cells of spinal cord is large number of Nissl bodies, which under transmission electron microscope are seen to be rough-surfaced endoplasmic reticulum free ribosomes (PALAY PALADE, 1955). The cell bodies of motor neurons are readily identified by ir large size, characteristic nucleus cytoplasm, synaptic apparatus (BODIAN, 1964), According to present observation, parts of cytoplasm which correspond to Nissl bodies are denser than cytoplasm surrounding m. They correspond to ribosomes (TANAKA, 1972), linear structures observed in Nissl bodies may be equivalent to rough-surfaced endoplasmic reticulum. The vesicles, cisterns membranous structures near nucleus can be regarded as Golgi complex. Mitochondria ir cristae are clearly identified under scanning electron microscope, though two membranes of mitochondria, outer inner, are not clear. On or h, no neurofilaments neurotubles could be identified. Secondly, as for myelinated nerve fibers under scanning electron microscope, myelin sheaths are observed in three dimensions consisting of numerous Fig. 8. Free surface of central canal. Cilia are uniform in distribution number from cell to

8 412 Fig. 9. K. OHTSUKI: A scanning electron micrograph for showing each cilia of central cell. 18,000 canal. They number 12-15

9 Scanning Electron Microscopy of Spinal Cord 413 membranes surrounding axon as a whirl. Generally, when membranes appear wavy, y are to be considered as artificial products caused by drying. The appearance of fibrils which are supposed to be collagen fibers lying on external sheath of axon resembles that of fibrils in schematic diagram of a myelinated nerve fiber by MORAN (1953). The myelin sheaths are also observed to extend branching processes which, according to present observation, often can be followed to cell body of a glial cell. BUNGE et al. (1961) have reported that several sheaths may be connected to one glial cell by processes. The processes in question are thus believed to be cytoplasmic processes of glial cells which connect glial cells myelin sheaths. Finally neuroglial cells are dealt with. On basis of light microscopic investigations, neuroglial cells of central nervous system are generally classified into four kinds, astrocytes, oligodendrocytes, microglia ependymal cells. However, identification of different neuroglial cells in transmission electron microscope preparations is not always easy except for ependymal cell. By scanning electron microscopy, it is also impossidle to make exact identification of different neuroglial cells, though ependymal cells are readily identified because y are arranged around central canal of spinal cord provided with cilia microvilli. As for ependymal tissue in ventricle of brain, comprehensive studies by transmission electron microscopy have been performed by BRIGHTMAN PALAY (1963), KOHNO USUI (1966). They have reported that re is a great difference in distribution number of cilia from cell to cell, numerous projections are found which are too irregular in ir dimensions to be termed microvilli. With regard to ependymal cells of central canal, SCHULTZ et al. (1956) have observed that ir free surface has two types of processes, a bulbous cytoplasmic expansion with many fingerlike microvilli true typical cilia. Both processes may be found on same ependymal cell. The present scanning electron micrographs demonstrate cilia of ependymal cells life-like in three dimensions. With regard to ciliary activity, it has been recognized up to date that direction of ciliary motion in brain corresponds to movement of cerebrospinal fluid. The rapid sweeping strokes seem to be coordinated in a particular direction, are thought to be important for circulation of cerebrospinal fluid in brain (WORTHINGTON CATHCART, 1963; DALEN et al., 1971). The cilia of central canal may be considered to have same motions as cilia in brain. In present observation, cilia in central canal are fixed in a particular direction, but it is not clear from this finding wher re is a correlation between ciliary movements circulation of cerebrospinal fluid in central canal. Acknowledgement. Grateful acknowledgement is made to Prof. Keiichi TANAKA for his constant guidance, to Mr. Y. KASHIMA Mr. H. OSATAKE for ir assistance in this study.

10 414 K. OHTSUKI: References Anderson, T. F.: Techniques for preservation of threedimensional structure in preparing specimens for electron microscope. Trans. N. Y. Acad. Sci. 13: (1951). Bodian, D.: An electron microscopic study of monkey spinal cord. Bull. Johns Hopkins Hosp. 114: (1964). Boyde, A., D. W. James, R. L. Tresman R. A. Willis: Outgrowth from chick embryo spinal cord in vitro, studied with scanning electron microscope. Z. Zellforsch. 90: 1-18 (1968). Brightman, M. W. S. L. Palay: The fine structure of ependyma in brain of rat. J. Cell Biol. 19: (1963). Bunge, R. P., M. B. Bunge H. Ris: Ultrastructural study of remyelination in an experimental lesion in adult cat spinal cord. J. biophys. biochem. Cytol. 10: (1961). Dalen, H., W. T. Schlapfer A. Mamoon: Cilia on cultured ependymal cells examined by scanning electron microscopy. Exp. Cell Res. 67: (1971). Hamberger, A., H. A. Hansson J. Sjostr: Surface structure of isolated neurons. Detachment of nerve terminals during axon regeneration. J. Cell Biol. 47: (1970). Kohno, K. T. Usui: Electron microscopic studies on ependymal cilia ir basal feet on ventral stalk of rat subfornical organ. Bull. Tokyo Med. Dent. Univ. 13: (1966). Fernez-Moran, H.: Sheath axon structures in internode portion of vertebrate mylinated nerve fibers. An electron microscope study of rat frog sciatic nerves. Exp. Cell Res. 1: (1950). Palay, S. L. G. E. Palade: The fine structure of neurons. J. biophys. biochem. Cytol. 1: (1955).

11 Scanning Electron Microscopy of Spinal Cord 415 Schultz, R., E. C. Berkowitz D. C. Pease: The electron microscopy of lamprey spinal cord. J. Morphol. 98: (1956). Spencer, P. S. A. R. Lieberman: Scanning electron microscopy of isolated peripheral nerve fibers. Normal surface structure alterations proximal to neuromas. Z. Zellforsch. 119: (1971). Tanaka, K.: Freezed resin cracking method for scanning electron microscopy of biological materials. Naturwiss. 59: 77 (1972). Worthington, W. C., Jr. R. S. Cathcart, III.: Ependymal cilia: Distribution activity in adult human brain. Science 139: (1963). Dr. Kenji OHTSUKI Department of Anatomy Tottori University School of Medicine Yonago, 683 Japan